Methods, Compositions, and Kits Comprising Linker Probes for Quantifying Polynucleotides

ABSTRACT

The present invention is directed to methods, reagents, kits, and compositions for identifying and quantifying target polynucleotide sequences. A linker probe comprising a 3′ target specific portion, a loop, and a stem is hybridized to a target polynucleotide and extended to form a reaction product that includes a reverse primer portion and the stem nucleotides. A detector probe, a specific forward primer, and a reverse primer can be employed in an amplification reaction wherein the detector probe can detect the amplified target polynucleotide based on the stem nucleotides introduced by the linker probe. In some embodiments a plurality of short miRNAs are queried with a plurality of linker probes, wherein the linker probes all comprise a universal reverse primer portion a different 3′ target specific portion and different stems. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application60/575,661, filed May 28, 2004, for “Methods, Compositions, and Kits forQuantifying Target Polynucleotides” by Chen and Zhou.

FIELD

The present teachings are in the field of molecular and cell biology,specifically in the field of detecting target polynucleotides such asmiRNA.

INTRODUCTION

RNA interference (RNAi) is a highly coordinated, sequence-specificmechanism involved in posttranscriptional gene regulation. During theinitial steps of process, a ribonuclease (RNase) II-like enzyme calledDicer reduces long double-strand RNA (dsRNA) and complex hairpinprecursors into: 1) small interfering RNAs (siRNA) that degrademessenger RNA (mRNA) and 2) micro RNAs (miRNAs) that can target mRNAsfor cleavage or attenuate. translation.

The siRNA class of molecules is thought to be comprised of 21-23nucleotide (nt) depluxes with characteristic dinucleotide 3′ overhangs(Ambros et al., 2003, RNA, 9 (3), 277-279). siRNA has been shown to actas the functional intermediate in RNAi, specifically directing cleavageof complementary mRNA targets in a process that is commonly regarded tobe an antiviral cellular defense mechanism (Elbashir et al., 2001,Nature, 411:6836), 494-498, Elbashir et al., 2001, Genes andDevelopment, 15 (2), 188-200). Target RNA cleavage is catalyzed by theRNA-induced silencing complex (RISC), which functions as a sRNA directedendonuclease (reviewed in Bartel, 2004, Cell, 116 (2), 281-297).

Micro RNAs (miRNAs) typically comprise single-stranded, endogenousoligoribonucleotides of roughly 22 (18-25) bases in length that areprocessed from larger stem-looped precursor RNAs. The first genesrecognized to encode miRNAs, lin-4 and let-7 of C. elegans, wereidentified on the basis of the developmental timing defects associatedwith the loss-of-function mutations (Lee et al., 1993, Cell, 75 (5),843-854; Reinhart et al., 2000, Nature, 403, (6772), 901-906; reviewedby Pasquinelli et al., 2002, Annual Review of Cell and DevelopmentalBiology, 18, 495-513). The breadth and importance of miRNA-directed generegulation are coming into focus as more miRNAs and regulatory targetsand functions are discovered. To date, a total of at least 700 miRNAshave been identified in C. elegans, Drosophila (Fire et al., 1998,Nature, 391 (6669(, 805-811), mouse, human (Lagos-Quintana et al., 2001,Science, 294 (5543), 853-858), and plants (Reinhart et al., 2002, Genesand Development, 16 (13), 1616-1626). Their sequences are typicallyconserved among different species. Size ranges from 18 to 25 nucleotidesfor miRNAs are the most commonly observed to date.

The function of most miRNAs is not known. Recently discovered miRNAfunctions include control of cell proliferation, cell death, and fatmetabolism in flies (Brennecke et al., 2003, cell, 113 (1), 25-36; Xu etal, 2003, Current Biology, 13 (9), 790-795), neuronal patterning innematodes (Johnston and Hobert, 2003, Nature, 426 (6968), 845-849),modulation of hematopoietic lineage differentiation in mammals (Chen etal., 2004, Science, 303 (5654), 83-87), and control of leaf and flowerdevelopment in plants (Aukerman and Sakai, 2003, Plant Cell, 15 (11),2730-2741; Chen, 2003, Science, 303 (5666):2022-2025; Emery et al.,2003, Current Biology, 13 (20), 1768-1774; Palatnik et al., 2003,Nature, 425 (6955), 257-263). There is speculation that miRNAs mayrepresent a new aspect of gene regulation.

Most miRNAs have been discovered by cloning. There are few cloning kitsavailable for researchers from Ambion and QIAGEN etc. The process islaborious and less accurate. Further, there has been little reliabletechnology available for miRNA quantitation (Allawi et al., Third WaveTechnologies, RNA. 2004 July; 10(7):1153-61). Northern blotting has beenused but results are not quantitative (Lagos-Quitana et al., 2001,Science, 294 (5543), 853-854). Many miRNA researchers are interested inmonitoring the level of the miRNAs at different tissues, at thedifferent stages of development, or after treatment with variouschemical agents. However, the short length of miRNAs has their studydifficult.

SUMMARY

In some embodiments, the present teachings provide a method fordetecting a micro RNA (miRNA) comprising; hybridizing the miRNA and alinker probe, wherein the linker probe comprises a stem, a loop, and a3′ target-specific portion, wherein the 3′ target-specific portion basepairs with the 3′ end region of the miRNA; extending the linker probe toform an extension reaction product; amplifying the extension reactionproduct to form an amplification product; and, detecting the miRNA.

In some embodiments, the present teachings provide a method fordetecting a target polynucleotide comprising; hybridizing the targetpolynucleotide and a linker probe, wherein the linker probe comprises astem, a loop, and a 3′ target-specific portion, wherein the 3′target-specific portion base pairs with the 3′ end region of the targetpolynucleotide; extending the linker probe to form an extension reactionproduct; amplifying the extension reaction product to form anamplification product in the presence of a detector probe, wherein thedetector probe comprises a nucleotide of the linker probe stem in theamplification product or a nucleotide of the linker probe stemcomplement in the amplification product; and, detecting the targetpolynucleotide.

In some embodiments, the present teachings provide a method fordetecting a miRNA molecule comprising; hybridizing the miRNA moleculeand a linker probe, wherein the linker probe comprises a stem, a loop,and a 3′ target specific portion, wherein the 3′ target-specific portionbase pairs with the 3′ end region of the target polynucleotide;extending the linker probe to form an extension reaction product;amplifying the extension reaction product in the presence of a detectorprobe to form an amplification product, wherein the detector probecomprises a nucleotide of the linker probe stem in the amplificationproduct or a nucleotide of the linker probe stem complement in theamplification product, and the detector probe further comprises anucleotide of the 3′ end region of the miRNA in the amplificationproduct or a nucleotide of the 3′ end region of the miRNA complement inthe amplification product; and, detecting the miRNA molecule.

In some embodiments, the present teachings provide a method fordetecting two different miRNAs from a single hybridization reactioncomprising; hybridizing a first miRNA and a first linker probe, and asecond miRNA and a second linker probe, wherein the first linker probeand the second linker probe each comprise a loop, a stem, and a 3′target-specific portion, wherein the 3′ target-specific portion of thefirst linker probe base pairs with the 3′ end region of the first miRNA,and wherein the 3′ target-specific portion of the second linker probebase pairs with the 3′ end region of the second miRNA; extending thefirst linker probe and the second linker probe to form extensionreaction products; dividing the extension reaction products into a firstamplification reaction to form a first amplification reaction product,and a second amplification reaction to form a second amplificationreaction product, wherein a primer in the first amplification reactioncorresponds with the first miRNA and not the second miRNA, and a primerin the second amplification reaction corresponds with the second miRNAand not the first miRNA, wherein a first detector probe in the firstamplification reaction differs from a second detector probe in thesecond amplification reaction, wherein the first detector probecomprises a nucleotide of the first linker probe stem of theamplification product or a nucleotide of the first linker probe stemcomplement in the first amplification product, wherein the seconddetector probe comprises a nucleotide of the second linker probe stem ofthe amplification product or a nucleotide of the second linker probestem complement in the amplification product; and, detecting the twodifferent miRNAs.

In some embodiments, the present teachings provide a method fordetecting two different target polynucleotides from a singlehybridization reaction comprising; hybridizing a first targetpolynucleotide and a first linker probe, and a second targetpolynucleotide and a second linker probe, wherein the first linker probeand the second linker probe each comprise a loop, a stem, and a 3′target-specific portion, wherein the 3′ target-specific portion of thefirst linker probe base pairs with the 3′ end region of the first targetpolynucleotide, and wherein the 3′ target-specific portion of the secondlinker probe base pairs with the 3′ end region of the second targetpolynucleotide; extending the first linker probe and the second linkerprobe to form extension reaction products; dividing the extensionreaction products into a first amplification reaction to form a firstamplification reaction product and a second amplification reaction toform a second amplification reaction product; and, detecting the twodifferent miRNA molecules.

In some embodiments, the present teachings provide a method fordetecting a miRNA molecule from a cell lysate comprising; hybridizingthe miRNA molecule from the cell lysate with a linker probe, wherein thelinker probe comprises a stem, a loop, and a 3′ target specific portion,wherein the 3′ target-specific portion base pairs with the 3′ end regionof the miRNA; extending the linker probe to form an extension reactionproduct; amplifying the extension reaction product to form anamplification product in the presence of a detector probe, wherein thedetector probe comprises a nucleotide of the linker probe stem of theamplification product or a nucleotide of the linker probe stemcomplement in the amplification product, and the detector probe furthercomprises a nucleotide of the 3′ end region of the miRNA in theamplification product or a nucleotide of the 3′ end region of the miRNAcomplement in the amplification product; and, detecting the miRNAmolecule.

A kit comprising; a reverse transcriptase and a linker probe, whereinthe linker probe comprises a stern, a loop, and a 3′ target-specificportion, wherein the 3′ target-specific portion corresponds to a miRNA.

The present teachings contemplate method for detecting a miRNA moleculecomprising a step of hybridizing, a step of extending, a step ofamplifying, and a step of detecting.

These and other features of the present teachings are set forth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 depicts certain aspects of various compositions according to someembodiments of the present teachings.

FIG. 2 depicts certain aspects of various compositions according to someembodiments of the present teachings.

FIG. 3 depicts certain sequences of various compositions according tosome embodiments of the present teachings.

FIG. 4 depicts one single-plex assay design according to someembodiments of the present teachings.

FIG. 5 depicts an overview of a multiplex assay design according to someembodiments of the present teachings.

FIG. 6 depicts a multiplex assay design according to some embodiments ofthe present teachings.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Aspects of the present teachings may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way. The section headings usedherein are for organizational purposes only and are not to be construedas limiting the described subject matter in any way. All literature andsimilar materials cited in this application, including but not limitedto, patents, patent applications, articles, books, treatises, andinternet web pages are expressly incorporated by reference in theirentirety for any purpose. When definitions of terms in incorporatedreferences appear to differ from the definitions provided in the presentteachings, the definition provided in the present teachings shallcontrol. It will be appreciated that there is an implied “about” priorto the temperatures, concentrations, times, etc discussed in the presentteachings, such that slight and insubstantial deviations are within thescope of the present teachings herein. In this application, the use ofthe singular includes the plural unless specifically stated otherwise.For example, “a primer” means that more than one primer can, but neednot, be present; for example but without limitation, one or more copiesof a particular primer species, as well as one or more versions of aparticular primer type, for example but not limited to, a multiplicityof different forward primers. Also, the use of “comprise”, “comprises”,“comprising”, “contain”, “contains”, “containing”, “include”,“includes”, and “including” are not intended to be limiting. It is to beunderstood that both the foregoing general description and the followingdetailed description are exemplary and explanatory only and are notrestrictive of the invention.

SOME DEFINITIONS

As used herein, the term “target polynucleotide” refers to apolynucleotide sequence that is sought to be detected. The targetpolynucleotide can be obtained from any source, and can comprise anynumber of different compositional components. For example, the targetcan be nucleic acid (e.g. DNA or RNA), transfer RNA, siRNA, and cancomprise nucleic acid analogs or other nucleic acid mimic. The targetcan be methylated, non-methylated, or both. The target can bebisulfite-treated and non-methylated cytosines converted to uracil.Further, it will be appreciated that “target polynucleotide” can referto the target polynucleotide itself, as well as surrogates thereof, forexample amplification products, and native sequences. In someembodiments, the target polynucleotide is a miRNA molecule. In someembodiments, the target polynucleotide lacks a poly-A tail. In someembodiments, the target polynucleotide is a short DNA molecule derivedfrom a degraded source, such as can be found in for example but notlimited to forensics samples (see for example Butler, 2001, Forensic DNATyping: Biology and Technology Behind STR Markers. The targetpolynucleotides of the present teachings can be derived from any of anumber of sources, including without limitation, viruses, prokaryotes,eukaryotes, for example but not limited to plants, fungi, and animals.These sources may include, but are not limited to, whole blood, a tissuebiopsy, lymph, bone marrow, amniotic fluid, hair, skin, semen,biowarfare agents, anal secretions, vaginal secretions, perspiration,saliva, buccal swabs, various environmental samples (for example,agricultural, water, and soil), research samples generally, purifiedsamples generally, cultured cells, and lysed cells. It will beappreciated that target polynucleotides can be isolated from samplesusing any of a variety of procedures known in the art, for example theApplied Biosystems ABI Prism™ 6100 Nucleic Acid PrepStation, and the ABIPrism™ 6700 Automated Nucleic Acid Workstation, Boom et al., U.S. Pat.No. 5,234,809, mirVana RNA isolation kit (Ambion), etc. It will beappreciated that target polynucleotides can be cut or sheared prior toanalysis, including the use of such procedures as mechanical force,sonication, restriction endonuclease cleavage, or any method known inthe art. In general, the target polynucleotides of the present teachingswill be single stranded, though in some embodiments the targetpolynucleotide can be double stranded, and a single strand can resultfrom denaturation.

As used herein, the term “3′ end region of the target polynucleotide”refers to the region of the target to which the 3′ target specificportion of the linker probe hybridizes. In some embodiments there can bea gap between the 3′ end region of the target polynucleotide and the 5′end of the linker probe, with extension reactions filling in the gap,though generally such scenarios are not preferred because of the likelydestabilizing effects on the duplex. In some embodiments, a miRNAmolecule is the target, in which case the term “3′ end region of themiRNA” is used.

As used herein, the term “linker probe” refers to a molecule comprisinga 3′ target specific portion, a stem, and a loop. Illustrative linkerprobes are depicted in FIG. 2 and elsewhere in the present teachings. Itwill be appreciated that the linker probes, as well as the primers ofthe present teachings, can be comprised of ribonucleotides,deoxynucleotides, modified ribonucleotides, modifieddeoxyribonucleotides, modified phosphate-sugar-backboneoligonucleotides, nucleotide analogs, or combinations thereof. For someillustrative teachings of various nucleotide analogs etc, see Fasman,1989, Practical Handbook of Biochemistry and Molecular Biology, pp.385-394, CRC Press, Boca Raton, Fla., Loakes, N.A.R. 2001, vol29:2437-2447, and Pellestor et al., Int J Mol Med. 2004 April;13(4):521-5.), references cited therein, and recent articles citingthese reviews. It will be appreciated that the selection of the linkerprobes to query a given target polynucleotide sequence, and theselection of which collection of target polynucleotide sequences toquery in a given reaction with which collection of linker probes, willinvolve procedures generally known in the art, and can involve the useof algorithms to select for those sequences with minimal secondary andtertiary structure, those targets with minimal sequence redundancy withother regions of the genome, those target regions with desirablethermodynamic characteristics, and other parameters desirable for thecontext at hand.

As used herein, the term “3′ target-specific portion” refers to thesingle stranded portion of a linker probe that is complementary to atarget polynucleotide. The 3′ target-specific portion is locateddownstream from the stem of the linker probe. Generally, the 3′target-specific portion is between 6 and 8 nucleotides long. In someembodiments, the 3′ target-specific portion is 7 nucleotides long. Itwill be appreciated that routine experimentation can produce otherlengths, and that 3′ target-specific portions that are longer than 8nucleotides or shorter than 6 nucleotides are also contemplated by thepresent teachings. Generally, the 3′-most nucleotides of the 3′target-specific portion should have minimal complementarity overlap, orno overlap at all, with the 3′ nucleotides of the forward primer; itwill be appreciated that overlap in these regions can produce undesiredprimer dimer amplification products in subsequent amplificationreactions. In some embodiments, the overlap between the 3′-mostnucleotides of the 3′ target-specific portion and the 3′ nucleotides ofthe forward primer is 0, 1, 2, or 3 nucleotides. In some embodiments,greater than 3 nucleotides can be complementary between the 3′-mostnucleotides of the 3′ target-specific portion and the 3′ nucleotides ofthe forward primer, but generally such scenarios will be accompanied byadditional non-complementary nucleotides interspersed therein. In someembodiments, modified bases such as. LNA can be used in the 3′ targetspecific portion to increase the Tm of the linker probe (see for examplePetersen et al., Trends in Biochemistry (2003), 21:2:74-81). In someembodiments, universal bases can be used, for example to allow forsmaller libraries of linker probes. Universal bases can also be used inthe 3′ target specific portion to allow for the detection of unknowntargets. For some descriptions of universal bases, see for exampleLoakes et al., Nucleic Acids Research, 2001, Volume 29, No. 12,2437-2447. In some embodiments, modifications including but not limitedto LNAs and universal bases can improve reverse transcriptionspecificity and potentially enhance detection specificity.

As used herein, the term “stem” refers to the double stranded region ofthe linker probe that is between the 3′ target-specific portion and theloop. Generally, the stem is between 6 and 20 nucleotides long (that is,6-20 complementary pairs of nucleotides, for a total of 12-40 distinctnucleotides). In some embodiments, the stem is 8-14 nucleotides long. Asa general matter, in those embodiments in which a portion of thedetector probe is encoded in the stem, the stem can be longer. In thoseembodiments in which a portion of the detector probe is not encoded inthe stem, the stem can be shorter. Those in the art will appreciate thatstems shorter that 6 nucleotides and longer than 20 nucleotides can beidentified in the course of routine methodology and without undueexperimentation, and that such shorter and longer stems are contemplatedby the present teachings. In some embodiments, the stem can comprise anidentifying portion.

As used herein, the term “loop” refers to a region of the linker probethat is located between the two complementary strands of the stern, asdepicted in FIG. 1 and elsewhere in the present teachings. Typically,the loop comprises single stranded nucleotides, though other moietiesmodified DNA or RNA, Carbon spacers such as C18, and/or PEG(polyethylene glycol) are also possible. Generally, the loop is between4 and 20 nucleotides long. In some embodiments, the loop is between 14and 18 nucleotides long. In some embodiments, the loop is 16 nucleotideslong. As a general matter, in those embodiments in which a reverseprimer is encoded in the loop, the loop can generally be longer. Inthose embodiments in which the reverse primer corresponds to both thetarget polynucleotide as well as the loop, the loop can generally beshorter. Those in the art will appreciate that loops shorter that 4nucleotides and longer than 20 nucleotides can be identified in thecourse of routine methodology and without undue experimentation, andthat such shorter and longer loops are contemplated by the presentteachings. In some embodiments, the loop can comprise an identifyingportion.

As used herein, the term “identifying portion” refers to a moiety ormoieties that can be used to identify a particular linker probe species,and as a result determine a target polynucleotide sequence, and canrefer to a variety of distinguishable moieties including zipcodes, aknown number of nucleobases, and combinations thereof. In someembodiments, an identifying portion, or an identifying portioncomplement, can hybridize to a detector probe, thereby allowingdetection of a target polynucleotide sequence in a decoding reaction.The terms “identifying portion complement” typically refers to at leastone oligonucleotide that comprises at least one sequence of nucleobasesthat are at least substantially complementary to and hybridize withtheir corresponding identifying portion. In some embodiments,identifying portion complements serve as capture moieties for attachingat least one identifier portion:element complex to at least onesubstrate; serve as “pull-out” sequences for bulk separation procedures;or both as capture moieties and as pull-out sequences (see for exampleO'Neil, et al., U.S. Pat. Nos. 6,638,760, 6,514,699, 6,146,511, and6,124,092). Typically, identifying portions and their correspondingidentifying portion complements are selected to minimize: internal,self-hybridization; cross-hybridization with different identifyingportion species, nucleotide sequences in a reaction composition,including but not limited to gDNA, different species of identifyingportion complements, or target-specific portions of probes, and thelike; but should be amenable to facile hybridization between theidentifying portion and its corresponding identifying portioncomplement. Identifying portion sequences and identifying portioncomplement sequences can be selected by any suitable method, for examplebut not limited to, computer algorithms such as described in PCTPublication Nos. WO 96/12014 and WO 96/41011 and in European PublicationNo. EP 799,897; and the algorithm and parameters of SantaLucia (Proc.Natl. Acad. Sci. 95:1460-65 (1998)). Descriptions of identifyingportions can be found in, among other places, U.S. Pat. Nos. 6,309,829(referred to as “tag segment” therein); 6,451,525 (referred to as “tagsegment” therein); 6,309,829 (referred to as “tag segment” therein);5,981,176 (referred to as “grid oligonucleotides” therein); 5,935,793(referred to as “identifier tags” therein); and PCT Publication No. WO01/92579 (referred to as “addressable support-specific sequences”therein). In some embodiments, the stem of the linker probe, the loop ofthe linker probe, or combinations thereof can comprise an identifyingportion, and the detector probe can hybridize to the correspondingidentifying portion. In some embodiments, the detector probe canhybridize to both the identifying portion as well as sequencecorresponding to the target polynucleotide. In some embodiments, atleast two identifying portion: identifying portion complement duplexeshave melting temperatures that fall within a Δ T_(m) range(T_(max)-T_(min)) of no more than 10° C. of each other. In someembodiments, at least two identifying portion: identifying portioncomplement duplexes have melting temperatures that fall within a Δ T_(m)range of 5° C. or less of each other. In some embodiments, at least twoidentifying portion: identifying portion complement duplexes havemelting temperatures that fall within a Δ T_(m range of) 2° C. or lessof each other. In some embodiments, at least one identifying portion orat least one identifying portion complement is used to separate theelement to which it is bound from at least one component of a ligationreaction composition, a digestion reaction composition, an amplifiedligation reaction composition, or the like. In some embodiments,identifying portions are used to attach at least one ligation product,at least one ligation product surrogate, or combinations thereof, to atleast one substrate. In some embodiments, at least one ligation product,at least one ligation product surrogate, or combinations thereof,comprise the same identifying portion. Examples of separation approachesinclude but are not limited to, separating a multiplicity of differentelement: identifying portion species using the same identifying portioncomplement, tethering a multiplicity of different element: identifyingportion species to a substrate comprising the same identifying portioncomplement, or both. In some embodiments, at least one identifyingportion complement comprises at least one label, at least one mobilitymodifier, at least one label binding portion, or combinations thereof.In some embodiments, at least one identifying portion complement isannealed to at least one corresponding identifying portion and,subsequently, at least part of that identifying portion complement isreleased and detected, see for example Published P.C.T. ApplicationWO04/4634 to Rosenblum et al., and Published P.C.T. ApplicationWO01/92579 to Wenz et al.,

As used herein, the term “extension reaction” refers to an elongationreaction in which the 3′ target specific portion of a linker probe isextended to form an extension reaction product comprising a strandcomplementary to the target polynucleotide. In some embodiments, thetarget polynucleotide is a miRNA molecule and the extension reaction isa reverse transcription reaction comprising a reverse transcriptase. Insome embodiments, the extension reaction is a reverse transcriptionreaction comprising a polymerase derived from a Eubacteria. In someembodiments, the extension reaction can comprise rTth polymerase, forexample as commercially available from Applied Biosystems catalog numberN808-0192, and N808-0098. In some embodiments, the target polynucleotideis a miRNA or other RNA molecule, and as such it will be appreciatedthat the use of polymerases that also comprise reverse transcriptionproperties can allow for some embodiments of the present teachings tocomprise a first reverse transcription reaction followed thereafter byan amplification reaction, thereby allowing for the consolidation of tworeactions in essentially a single reaction. In some embodiments, thetarget polynucleotide is a short DNA molecule and the extension reactioncomprises a polymerase and results in the synthesis of a 2^(nd) strandof DNA. In some embodiments, the consolidation of the extension reactionand a subsequent amplification reaction is further contemplated by thepresent teachings.

As used herein, the term “primer portion” refers to a region of apolynucleotide sequence that can serve directly, or by virtue of itscomplement, as the template upon which a primer can anneal for any of avariety of primer nucleotide extension reactions known in the art (forexample, PCR). It will be appreciated by those of skill in the art thatwhen two primer portions are present on a single polynucleotide, theorientation of the two primer portions is generally different. Forexample, one PCR primer can directly hybridize to a first primerportion, while the other PCR primer can hybridize to the complement ofthe second primer portion. In addition, “universal” primers and primerportions as used herein are generally chosen to be as unique as possiblegiven the particular assays and host genomes to ensure specificity ofthe assay.

As used herein, the term “forward primer” refers to a primer thatcomprises an extension reaction product portion and a tail portion. Theextension reaction product portion of the forward primer hybridizes tothe extension reaction product. Generally, the extension reactionproduct portion of the forward primer is between 9 and 19 nucleotides inlength. In some embodiments, the extension reaction product portion ofthe forward primer is 16 nucleotides. The tail portion is locatedupstream from the extension reaction product portion, and is notcomplementary with the extension reaction product; after a round ofamplification however, the tail portion can hybridize to complementarysequence of amplification products. Generally, the tail portion of theforward primer is between 5-8 nucleotides long. In some embodiments, thetail portion of the forward primer is 6 nucleotides long. Those in theart will appreciate that forward primer tail portion lengths shorterthan 5 nucleotides and longer than 8 nucleotides can be identified inthe course of routine methodology and without undue experimentation, andthat such shorter and longer forward primer tail portion lengths arecontemplated by the present teachings. Further, those in the art willappreciate that lengths of the extension reaction product portion of theforward primer shorter than 9 nucleotides in length and longer than 19nucleotides in length can be identified in the course of routinemethodology and without undue experimentation, and that such shorter andlonger extension reaction product portion of forward primers arecontemplated by the present teachings.

As used herein, the term “reverse primer” refers to a primer that whenextended forms a strand complementary to the target polynucleotide. Insome embodiments, the reverse primer corresponds with a region of theloop of the linker probe. Following the extension reaction, the forwardprimer can be extended to form a second strand product. The reverseprimer hybridizes with this second strand product, and can be extendedto continue the amplification reaction. In some embodiments, the reverseprimer corresponds with a region of the loop of the linker probe, aregion of the stem of the linker probe, a region of the targetpolynucleotide, or combinations thereof. Generally, the reverse primeris between 13-16 nucleotides long. In some embodiments the reverseprimer is 14 nucleotides long. In some embodiments, the reverse primercan further comprise a non-complementary tail region, though such a tailis not required. In some embodiments, the reverse primer is a “universalreverse primer,” which indicates that the sequence of the reverse primercan be used in a plurality of different reactions querying differenttarget polynucleotides, but that the reverse primer nonetheless is thesame sequence.

The term “upstream” as used herein takes on its customary meaning inmolecular biology, and refers to the location of a region of apolynucleotide that is on the 5′ side of a “downstream” region.Correspondingly, the term “downstream” refers to the location of aregion of a polynucleotide that is on the 3′ side of an “upstream”region.

As used herein, the term “hybridization” refers to the complementarybase-pairing interaction of one nucleic acid with another nucleic acidthat results in formation of a duplex, triplex, or other higher-orderedstructure, and is used herein interchangeably with “annealing.”Typically, the primary interaction is base specific, e.g., A/T and G/C,by Watson/Crick and Hoogsteen-type hydrogen bonding. Base-stacking andhydrophobic interactions can also contribute to duplex stability.Conditions for hybridizing detector probes and primers to complementaryand substantially complementary target sequences are well known, e.g.,as described in Nucleic Acid Hybridization, A Practical Approach, B.Hames and S. Higgins, eds., IRL Press, Washington, D.C. (1985) and J.Wetmur and N. Davidson, Mol. Biol. 31:349 et seq. (1968). In general,whether such annealing takes place is influenced by, among other things,the length of the polynucleotides and the complementary, the pH, thetemperature, the presence of mono- and divalent cations, the proportionof G and C nucleotides in the hybridizing region, the viscosity of themedium, and the presence of denaturants. Such variables influence thetime required for hybridization. Thus, the preferred annealingconditions will depend upon the particular application. Such conditions,however, can be routinely determined by the person of ordinary skill inthe art without undue experimentation. It will be appreciated thatcomplementarity need not be perfect; there can be a small number of basepair mismatches that will minimally interfere with hybridization betweenthe target sequence and the single stranded nucleic acids of the presentteachings. However, if the number of base pair mismatches is so greatthat no hybridization can occur under minimally stringent conditionsthen the sequence is generally not a complementary target sequence.Thus, complementarity herein is meant that the probes or primers aresufficiently complementary to the target sequence to hybridize under theselected reaction conditions to achieve the ends of the presentteachings.

As used herein, the term “amplifying” refers to any means by which atleast a part of a target polynucleotide, target polynucleotidesurrogate, or combinations thereof, is reproduced, typically in atemplate-dependent manner, including without limitation, a broad rangeof techniques for amplifying nucleic acid sequences, either linearly orexponentially. Exemplary means for performing an amplifying step includeligase chain reaction (LCR), ligase detection reaction (LDR), ligationfollowed by Q-replicase amplification, PCR, primer extension, stranddisplacement amplification (SDA), hyperbranched strand displacementamplification, multiple displacement amplification (MDA), nucleic acidstrand-based amplification (NASBA), two-step multiplexed amplifications,rolling circle amplification (RCA) and the like, including multiplexversions or combinations thereof, for example but not limited to,OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (alsoknown as combined chain reaction—CCR), and the like. Descriptions ofsuch techniques can be found in, among other places, Sambrook et al.Molecular Cloning, 3^(rd) Edition, Ausbel et al.; PCR Primer: ALaboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); TheElectronic Protocol Book, Chang Bioscience (2002), Msuih et al., J.Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R.Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson et al., CurrOpin Biotechnol. 1993 February; 4(1):41-7, U.S. Pat. No. 6,027,998; U.S.Pat. No. 6,605,451, Barany et al., PCT Publication No. WO 97/31256; Wenzet al., PCT Publication No. WO 01/92579; Day et al., Genomics, 29(1):152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis etal., PCR Protocols: A Guide to Methods and Applications, Academic Press(1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenauet al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin,Development of a Multiplex Ligation Detection Reaction DNA Typing Assay,Sixth International Symposium on Human Identification, 1995 (availableon the world wide web at:promega.com/geneticidproc/ussymp6proc/blegrad.html); LCR Kit InstructionManual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc.Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res.25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999);Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany andGelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res. 20:1691-96(1992); Polstra et al., BMC Inf. Dis. 2:18-(2002); Lage et al., GenomeRes. 2003 February; 13(2):294-307, and Landegren et al., Science241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 November;2(6):542-8., Cook et al., J Microbiol Methods. 2003 May; 53(2):165-74,Schweitzer et al., Curr Opin Biotechnol. 2001 February; 12(1):21-7, U.S.Pat. No. 5,830,711, U.S. Pat. No. 6,027,889, U.S. Pat. No. 5,686,243,Published P.C.T. Application WO0056927A3, and Published P.C.T.Application WO9803673A1. In some embodiments, newly-formed nucleic acidduplexes are not initially denatured, but are used in theirdouble-stranded form in one or more subsequent steps. An extensionreaction is an amplifying technique that comprises elongating a linkerprobe that is annealed to a template in the 5′ to 3′ direction using anamplifying means such as a polymerase and/or reverse transcriptase.According to some embodiments, with appropriate buffers, salts, pH,temperature, and nucleotide triphosphates, including analogs thereof,i.e., under appropriate conditions, a polymerase incorporatesnucleotides complementary to the template strand starting at the 3′-endof an annealed linker probe, to generate a complementary strand. In someembodiments, the polymerase used for extension lacks or substantiallylacks 5′ exonuclease activity. In some embodiments of the presentteachings, unconventional nucleotide bases can be introduced into theamplification reaction products and the products treated by enzymatic(e.g., glycosylases) and/or physical-chemical means in order to renderthe product incapable of acting as a template for subsequentamplifications. In some embodiments, uracil can be included as anucleobase in the reaction mixture, thereby allowing for subsequentreactions to decontaminate carryover of previous uracil-containingproducts by the use of uracil-N-glycosylase (see for example PublishedP.C.T. Application WO9201814A2). In some embodiments of the presentteachings, any of a variety of techniques can be employed prior toamplification in order to facilitate amplification success, as describedfor example in Radstrom et al., Mol Biotechnol. 2004 February;26(2):133-46. In some embodiments, amplification can be achieved in aself-contained integrated approach comprising sample preparation anddetection, as described for example in U.S. Pat. Nos. 6,153,425 and6,649,378. Reversibly modified enzymes, for example but not limited tothose described in U.S. Pat. No. 5,773,258, are also within the scope ofthe disclosed teachings. The present teachings also contemplate variousuracil-based decontamination strategies, wherein for example uracil canbe incorporated into an amplification reaction, and subsequentcarry-over products removed with various glycosylase treatments (see forexample U.S. Pat. No. 5,536,649, and U.S. Provisional Application60/584,682 to Andersen et al.,). Those in the art will understand thatany protein with the desired enzymatic activity can be used in thedisclosed methods and kits. Descriptions of DNA polymerases, includingreverse transcriptases, uracil N-glycosylase, and the like, can be foundin, among other places, Twyman, Advanced Molecular Biology, BIOSScientific Publishers, 1999; Enzyme Resource Guide, rev. 092298,Promega, 1998; Sambrook and Russell; Sambrook et al.; Lehninger; PCR:The Basics; and Ausbel et al.

As used herein, the term “detector probe” refers to a molecule used inan amplification reaction, typically for quantitative or real-time PCRanalysis, as well as end-point analysis. Such detector probes can beused to monitor the amplification of the target polynucleotide. In someembodiments, detector probes present in an amplification reaction aresuitable for monitoring the amount of amplicon(s) produced as a functionof time. Such detector probes include, but are not limited to, the5′-exonuclease assay (TaqMan® probes described herein (see also U.S.Pat. No. 5,538,848) various stem-loop molecular beacons (see e.g., U.S.Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, 1996, NatureBiotechnology 14:303-308), stemless or linear beacons (see, e.g., WO99/21881), PNA Molecular Beacons™ (see, e.g., U.S. Pat. Nos. 6,355,421and 6,593,091), linear PNA beacons (see, e.g., Kubista et al., 2001,SPIE 4264:53-58), non-FRET probes (see, e.g., U.S. Pat. No. 6,150,097),Sunrise ®/Amplifluor probes (U.S. Pat. No. 6,548,250), stem-loop andduplex Scorpion™ probes (Solinas et al., 2001, Nucleic Acids Research29:E96 and U.S. Pat. No. 6,589,743), bulge loop probes (U.S. Pat. No.6,590,091), pseudo knot probes (U.S. Pat. No. 6,589,250), cyclicons(U.S. Pat. No. 6,383,752), MGB Eclipse™ probe (Epoch Biosciences),hairpin probes (U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA)light-up probes, self-assembled nanoparticle probes, andferrocene-modified probes described, for example, in U.S. Pat. No.6,485,901; Mhlanga et al., 2001, Methods 25:463-471; Whitcombe et al.,1999, Nature Biotechnology. 17:804-807; Isacsson et al., 2000, MolecularCell Probes. 14:321-328; Svanvik et al., 2000, Anal Biochem. 281:26-35;Wolffs et al., 2001, Biotechniques 766:769-771; Tsourkas et al., 2002,Nucleic Acids Research. 30:4208-4215; Riccelli et al., 2002, NucleicAcids Research 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332;Maxwell et al., 2002, J. Am. Chem. Soc. 124:9606-9612; Broude et al.,2002, Trends Biotechnol. 20:249-56; Huang et al., 2002, Chem Res.Toxicol. 15:118-126; and Yu et al., 2001, J. Am. Chem. Soc14:11155-11161. Detector probes can also comprise quenchers, includingwithout limitation black hole quenchers (Biosearch), Iowa Black (IDT),QSY quencher (Molecular Probes), and Dabsyl and Dabcelsulfonate/carboxylate Quenchers (Epoch). Detector probes can alsocomprise two probes, wherein for example a fluor is on one probe, and aquencher is on the other probe, wherein hybridization of the two probestogether on a target quenches the signal, or wherein hybridization onthe target alters the signal signature via a change in fluorescence.Detector probes can also comprise sulfonate derivatives of fluorescenindyes with SO3 instead of the carboxylate group, phosphoramidite forms offluorescein, phosphoramidite forms of CY 5 (commercially available forexample from Amersham). In some embodiments, interchelating labels areused such as ethidium bromide, SYBR® Green I (Molecular Probes), andPicoGreen® (Molecular Probes), thereby allowing visualization inreal-time, or end point, of an amplification product in the absence of adetector probe. In some embodiments, real-time visualization cancomprise both an intercalating detector probe and a sequence-baseddetector probe can be employed. In some embodiments, the detector probeis at least partially quenched when not hybridized to a complementarysequence in the amplification reaction, and is at least partiallyunquenched when hybridized to a complementary sequence in theamplification reaction. In some embodiments, the detector probes of thepresent teachings have a Tm of 63-69 C, though it will be appreciatedthat guided by the present teachings routine experimentation can resultin detector probes with other Tms. In some embodiments, probes canfurther comprise various modifications such as a minor groove binder(see for example U.S. Pat. No. 6,486,308) to further provide desirablethermodynamic characteristics. In some embodiments, detector probes cancorrespond to identifying portions or identifying portion complements.

The term “corresponding” as used herein refers to a specificrelationship between the elements to which the term refers. Somenon-limiting examples of corresponding include: a linker probe cancorrespond with a target polynucleotide, and vice versa. A forwardprimer can correspond with a target polynucleotide, and vice versa. Alinker probe can correspond with a forward primer for a given targetpolynucleotide, and vice versa. The 3′ target-specific portion of thelinker probe can correspond with the 3′ region of a targetpolynucleotide, and vice versa. A detector probe can correspond with aparticular region of a target polynucleotide and vice versa. A detectorprobe can correspond with a particular identifying portion and viceversa. In some cases, the corresponding elements can be complementary.In some cases, the corresponding elements are not complementary to eachother, but one element can be complementary to the complement of anotherelement.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, the term “reaction vessel” generally refers to anycontainer in which a reaction can occur in accordance with the presentteachings. In some embodiments, a reaction vessel can be an eppendorftube, and other containers of the sort in common practice in modernmolecular biology laboratories. In some embodiments, a reaction vesselcan be a well in microtitre plate, a spot on a glass slide, or a well inan Applied Biosystems TaqMan Low Density Array for gene expression(formerly MicroCard™). For example, a plurality of reaction vessels canreside on the same support. In some embodiments, lab-on-a-chip likedevices, available for example from Caliper and Fluidgm, can provide forreaction vessels. In some embodiments, various microfluidic approachesas described in U.S. Provisional Application 60/545,674. to Wenz et al.,can be employed. It will be recognized that a variety of reaction vesselare available in the art and within the scope of the present teachings.

As used herein, the term “detection” refers to any of a variety of waysof determining the presence and/or quantity and/or identity of a targetpolynucleoteide. In some embodiments employing a donor moiety and signalmoiety, one may use certain energy-transfer fluorescent dyes. Certainnonlimiting exemplary pairs of donors (donor moieties) and acceptors(signal moieties) are illustrated, e.g., in U.S. Pat. Nos. 5,863,727;5,800,996; and 5,945,526. Use of some combinations of a donor and anacceptor have been called FRET (Fluorescent Resonance Energy Transfer).In some embodiments, fluorophores that can be used as signaling probesinclude, but are not limited to, rhodamine, cyanine 3 (Cy 3), cyanine 5(Cy 5), fluorescein, Vic™, Liz™, Tamra™, 5-Fam™ 6-Fam™ and Texas Red(Molecular Probes). (Vic™, Liz™, Tamra™ 5-Fam™ and 6-Fam™ (all availablefrom Applied Biosystems, Foster City, Calif.). In some embodiments, theamount of detector probe that gives a fluorescent signal in response toan excited light typically relates to the amount of nucleic acidproduced in the amplification reaction. Thus, in some embodiments, theamount of fluorescent signal is related to the amount of product createdin the amplification reaction. In such embodiments, one can thereforemeasure the amount of amplification product by measuring the intensityof the fluorescent signal from the fluorescent indicator. According tosome embodiments, one can employ an internal standard to quantify theamplification product indicated by the fluorescent signal. See, e.g.,U.S. Pat. No. 5,736,333. Devices have been developed that can perform athermal cycling reaction with compositions containing a fluorescentindicator, emit a light beam of a specified wavelength, read theintensity of the fluorescent dye, and display the intensity offluorescence after each cycle. Devices comprising a thermal cycler,light beam emitter, and a fluorescent signal detector, have beendescribed, e.g., in U.S. Pat. Nos. 5,928,907; 6,015,674; and 6,174,670,and include, but are not limited to the ABI Prism® 7700 SequenceDetection System (Applied Biosystems, Foster City, Calif.), the ABIGeneAmp® 5700 Sequence Detection System (Applied Biosystems, FosterCity, Calif.), the ABI GeneAmp® 7300 Sequence Detection System (AppliedBiosystems, Foster City, Calif.), and the ABI GeneAmp® 7500 SequenceDetection System (Applied Biosystems). In some embodiments, each ofthese functions can be performed by separate devices. For example, ifone employs a Q-beta replicase reaction for amplification, the reactionmay not take place in a thermal cycler, but could include a light beamemitted at a specific wavelength, detection of the fluorescent signal,and calculation and display of the amount of amplification product. Insome embodiments, combined thermal cycling and fluorescence detectingdevices can be used for precise quantification of target nucleic acidsequences in samples. In some embodiments, fluorescent signals can bedetected and displayed during and/or after one or more thermal cycles,thus permitting monitoring of amplification products as the reactionsoccur in “real time.” In some embodiments, one can use the amount ofamplification product and number of amplification cycles to calculatehow much of the target nucleic acid sequence was in the sample prior toamplification. In some embodiments, one could simply monitor the amountof amplification product after a predetermined number of cyclessufficient to indicate the presence of the target nucleic acid sequencein the sample. One skilled in the art can easily determine, for anygiven sample type, primer sequence, and reaction condition, how manycycles are sufficient to determine the presence of a given targetpolynucleotide. As used herein, determining the presence of a target cancomprise identifying it, as well as optionally quantifying it. In someembodiments, the amplification products can be scored as positive ornegative as soon as a given number of cycles is complete. In someembodiments, the results may be transmitted electronically directly to adatabase and tabulated. Thus, in some embodiments, large numbers ofsamples can be processed and analyzed with less time and labor when suchan instrument is used. In some embodiments, different detector probesmay distinguish between different target polynucleoteides. Anon-limiting example of such a probe is a 5′-nuclease fluorescent probe,such as a TaqMan® probe molecule, wherein a fluorescent molecule isattached to a fluorescence-quenching molecule through an oligonucleotidelink element. In some embodiments, the oligonucleotide link element ofthe 5′-nuclease fluorescent probe binds to a specific sequence of anidentifying portion or its complement. In some embodiments, different5′-nuclease fluorescent probes, each fluorescing at differentwavelengths, can distinguish between different amplification productswithin the same amplification reaction. For example, in someembodiments, one could use two different 5′-nuclease fluorescent probesthat fluoresce at two different wavelengths (WL_(A) and WL_(B)) and thatare specific to two different stem regions of two different extensionreaction products (A′ and B′, respectively). Amplification product A′ isformed if target nucleic acid sequence A is in the sample, andamplification product B′ is formed if target nucleic acid sequence B isin the sample. In some embodiments, amplification product A′ and/or B′may form even if the appropriate target nucleic acid sequence is not inthe sample, but such occurs to a measurably lesser extent than when theappropriate target nucleic acid sequence is in the sample. Afteramplification, one can determine which specific target nucleic acidsequences are present in the sample based on the wavelength of signaldetected and their intensity. Thus, if an appropriate detectable signalvalue of only wavelength WL_(A) is detected, one would know that thesample includes target nucleic acid sequence A, but not target nucleicacid sequence B. If an appropriate detectable signal value of bothwavelengths WL_(A) and WL_(B) are detected, one would know that thesample includes both target nucleic acid sequence A and target nucleicacid sequence B. In some embodiments, detection can occur through any ofa variety of mobility dependent analytical techniques based ondifferential rates of migration between different analyte species.Exemplary mobility-dependent analysis techniques includeelectrophoresis, chromatography, mass spectroscopy, sedimentation, e.g.,gradient centrifugation, field-flow fractionation, multi-stageextraction techniques, and the like. In some embodiments, mobilityprobes can be hybridized to amplification products, and the identity ofthe target polynucleotide determined via a mobility dependent analysistechnique of the eluted mobility probes, as described for example inPublished P.C.T. Application WO04/46344 to Rosenblum et al., andWO01/92579 to Wenz et al.,. In some embodiments, detection can beachieved by various microarrays and related software such as the AppliedBiosystems Array System with the Applied Biosystems 1700Chemiluminescent Microarray Analyzer and other commercially availablearray systems available from Affymetrix, Agilent, Illumina, and AmershamBiosciences, among others (see also Gerry et al., J. Mol. Biol.292:251-62, 1999; De Bellis et al., Minerva Biotec 14:247-52, 2002; andStears et al., Nat. Med. 9:140-45, including supplements, 2003). It willalso be appreciated that detection can comprise reporter groups that areincorporated into the reaction products, either as part of labeledprimers or due to the incorporation of labeled dNTPs during anamplification, or attached to reaction products, for example but notlimited to, via hybridization tag complements comprising reporter groupsor via linker arms that are integral or attached to reacton products.Detection of unlabeled reaction products, for example using massspectrometry, is also within the scope of the current teachings.

Exemplary Embodiments

FIG. 1 depicts certain compositions according to some embodiments of thepresent teachings. Top, a miRNA molecule (1, dashed line) is depicted.Middle, a linker probe (2) is depicted, illustrating a 3′ targetspecific portion (3), a stem (4), and a loop (5). Bottom, a miRNAhybridized to a linker probe is depicted, illustrating the 3′ targetspecific portion of the linker probe (3) hybridized to the 3′ end regionof the miRNA (6).

As shown in FIG. 2, a target polynucleotide (9, dotted line) isillustrated to show the relationship with various components of thelinker probe (10), the detector probe (7), and the reverse primer (8),according to various non-limiting embodiments of the present teachings.For example as shown in FIG. 2A, in some embodiments the detector probe(7) can correspond with the 3′ end region of the target polynucleotidein the amplification product as well as a region upstream from the 3′end region of the target polynucleotide in the amplification product.(Here, the detector probe is depicted as rectangle (7) with an F and aQ, symbolizing a TaqMan probe with a florophore (F) and a quencher (Q)).Also shown in FIG. 2A, the loop can correspond to the reverse primer(8). In some embodiments as shown in FIG. 2B, the detector probe (7) cancorrespond with a region of the amplification product corresponding withthe 3′ end region of the target polynucleotide in the amplificationproduct, as well as a region upstream from the 3′ end region of thetarget polynucleotide in the amplification product, as well as thelinker probe stem in the amplification product. Also shown in FIG. 2B,the upstream region of the stem, as well as the loop, can correspond tothe reverse primer (8). In some embodiments as shown in FIG. 2C, thedetector probe can correspond to the amplification product in a mannersimilar to that shown in FIG. 2B, but the loop can correspond to thereverse primer (8). In some embodiments as shown in FIG. 2D, thedetector probe (7) can correspond with the linker probe stem in theamplification product. Also shown in FIG. 2D, the upstream region of thestem, as well as the loop can correspond to the reverse primer (8). Itwill be appreciated that various related strategies for implementing thedifferent functional regions of these compositions are possible in lightof the present teachings, and that such derivations are routine to onehaving ordinary skill in the art without undue experimentation.

FIG. 3 depicts the nucleotide relationship for the micro RNA MiR-16(boxed, 11) according to some embodiments of the present teachings.Shown here is the interrelationship of MiR-16 to a forward primer (12),a linker probe (13), a TaqMan detector probe (14), and a reverse primer(boxed, 15). The TaqMan probe comprises a 3′ minor groove binder (MGB),and a 5′ FAM florophore. It will be appreciated that in some embodimentsof the present teachings the detector probes, such as for example TaqManprobes, can hybridize to either strand of an amplification product. Forexample, in some embodiments the detector probe can hybridize to thestrand of the amplification product corresponding to the first strandsynthesized. In some embodiments, the detector probe can hybridize tothe strand of the amplification product corresponding to the secondstrand synthesized.

FIG. 4 depicts a single-plex assay design according to some embodimentsof the present teachings. Here, a miRNA molecule (16) and a linker probe(17) are hybridized together (18). The 3′ end of the linker probe of thetarget-linker probe composition is extended to form an extension product(19) that can be amplified in a PCR. The PCR can comprise a miRNAspecific forward primer (20) and a reverse primer (21). The detection ofa detector probe (22) during the amplification allows for quantitationof the miRNA.

FIG. 5 depicts an overview of a multiplex assay design according to someembodiments of the present teachings. Here, a multiplexed hybridizationand extension reaction is performed in a first reaction vessel (23).Thereafter, aliquots of the extension reaction products from the firstreaction vessel are transferred into a plurality of amplificationreactions (here, depicted as PCRs 1, 2, and 3) in a plurality of secondreaction vessels. Each PCR can comprise a distinct primer pair and adistinct detector probe. In some embodiments, a distinct primer pair butthe same detector probe can be present in each of a plurality of PCRs.

FIG. 6 depicts a multiplex assay design according to some embodiments ofthe present teachings. Here, three different miRNAs (24, 25, and 26) arequeried in a hybridization reaction comprising three different linkerprobes (27, 28, and 29). Following hybridization and extension to formextension products (30, 31, and 32), the extension products are dividedinto three separate amplification reactions. (Though not explicitlyshown, it will be appreciated that a number of copies of the moleculesdepicted by 30, 31, and 32 can be present, such that each of the threeamplification reactions can have copies of 30, 31, and 32.) PCR 1comprises a forward primer specific for miRNA 24 (33), PCR 2 comprises aforward primer specific for miRNA 25 (34), and PCR 3 comprises a forwardprimer specific for miRNA 26 (35). Each of the forward primers furthercomprise a non-complementary tail portion. PCR 1, PCR 2, and PCR 3 allcomprise the same universal reverse primer 36. Further, PCR 1 comprisesa distinct detector probe (37) that corresponds to the 3′ end region ofmiRNA 24 and the stem of linker probe 27, PCR 2 comprises a distinctdetector probe (38) that corresponds to the 3′ end region of miRNA 25and the stem of linker probe 28, and PCR 3 comprises a distinct detectorprobe (39) that corresponds to the 3′ region of miRNA 26 and the stem oflinker probe 29.

The present teachings also contemplate reactions comprisingconfigurations other than a linker probe. For example, in someembodiments, two hybridized molecules with a sticky end can be employed,wherein for example an overlapping 3′ sticky end hybridizes with the 3′end region of the target polynucleotide. Some descriptions of twomolecule configurations that can be employed in the present teachingscan be found in Chen et al., U.S. Provisional Application 60/517,470.Viewed in light of the present teachings herein, one of skill in the artwill appreciate that the approaches of Chen et al., can also be employedto result in extension reaction products that are longer that the targetpolynucleotide. These longer products can be detected with detectorprobes by, for example, taking advantage of the additional nucleotidesintroduced into the reaction products.

The present teachings also contemplate embodiments wherein the linkerprobe is ligated to the target polynucleotide, as described for examplein Chen et al., U.S. Provisional Application 60/575,661, and thecorresponding co-filed U.S. Provisional application co-filed herewith

Further, it will be appreciated that in some embodiments of the presentteachings, the two molecule configurations in Chen et al., U.S.Provisional Application 60/517,470 can be applied in embodimentscomprising the linker approaches discussed in Chen et al., U.S.Provisional Application 60/575,661.

Generally however, the loop structure of the present teachings willenhance the Tm of the target polynucleotide-linker probe duplex. Withoutbeing limited to any particular theory, this enhanced Tm could possiblybe due to base stacking effects. Also, the characteristics of the loopedlinker probe of the present teachings can minimize nonspecific primingduring the extension reaction, and/or a subsequent amplificationreaction such as PCR. Further, the looped linker probe of the presentteachings can better differentiate mature and precursor forms of miRNA,as illustrated infra in Example 6.

The present teachings also contemplate encoding and decoding reactionschemes, wherein a first encoding extension reaction is followed by asecond decoding amplification reaction, as described for example inLivak et al., U.S. Provisional Application 60/556,162, Chen et al., U.S.Provisional Application 60/556,157, Andersen et al., U.S. ProvisionalApplication 60/556,224, and Lao et al., U.S. Provisional Application60/556,163.

The present teachings also contemplate a variety of strategies tominimize the number of different molecules in multiplexed amplificationstrategies, as described for example in Whitcombe et al., U.S. Pat. No.6,270,967.

In certain embodiments, the present teachings also provide kits designedto expedite performing certain methods. In some embodiments, kits serveto expedite the performance of the methods of interest by assembling twoor more components used in carrying out the methods. In someembodiments, kits may contain components in pre-measured unit amounts tominimize the need for measurements by end-users. In some embodiments,kits may include instructions for performing one or more methods of thepresent teachings. In certain embodiments, the kit components areoptimized to operate in conjunction with one another.

For example, the present teachings provide a kit comprising, a reversetranscriptase and a linker probe, wherein the linker probe comprises astem, a loop, and a 3′ target-specific portion, wherein the 3′target-specific portion corresponds to a miRNA. In some embodiments, thekits can comprise a DNA polymerase. In some embodiments, the kits cancomprise a primer pair. In some embodiments, the kits can furthercomprise a forward primer specific for a miRNA, and, a universal reverseprimer, wherein the universal reverse primer comprises a nucleotide ofthe loop of the linker probe. In some embodiments, the kits can comprisea plurality of primer pairs, wherein each primer pair is in one reactionvessel of a plurality of reaction vessels. In some embodiments, the kitscan comprise a detector probe. In some embodiments, the detector probecomprises a nucleotide of the linker probe stem in the amplificationproduct or a nucleotide of the linker probe stem complement in theamplification product, and the detector probe further comprises anucleotide of the 3′ end region of the miRNA in the amplificationproduct or a nucleotide of the 3′ end region of the miRNA complement inthe amplification product.

The present teachings further contemplate kits comprising a means forhybridizing, a means for extending, a means for amplifying, a means fordetecting, or combinations thereof.

While the present teachings have been described in terms of theseexemplary embodiments, the skilled artisan will readily understand thatnumerous variations and modifications of these exemplary embodiments arepossible without undue experimentation. All such variations andmodifications are within the scope of the current teachings. Aspects ofthe present teachings may be further understood in light of thefollowing examples, which should not be construed as limiting the scopeof the teachings in any way.

Example 1

A single-plex reaction was performed in replicate for a collection ofmouse miRNAs, and the effect of the presence or absence of ligase, aswell as the presence or absence of reverse transcriptase, determined.The results are shown in Table 1 as Ct values.

First, a 6 ul reaction was set up comprising: 1 ul Reverse TranscriptionEnzyme Mix (Applied Biosystems part number 4340444) (or 1 ul dH20), 0.5ul T4 DNA Ligase (400 units/ul, NEB) (or 0.5 ul dH20), 0.25 ul 2M KCl,0.05 ul dNTPs (25 mM each), 0.25 ul T4 Kinase (10 units/ul, NEB), 1 ul10X T4 DNA ligase buffer (NEB), 0.25 ul Applied Biosystems RNaseInhibitor (10 units/ul), and 2.2 ul dH20 Next, 2 ul of the linker probe(0.25 uM) and RNA samples (2 ul of 0.25 ug/ul mouse lung total RNA(Ambion, product number 7818) were added. Next, the reaction was mixed,spun briefly, and placed on ice for 5 minutes.

The reaction was then incubated at 16 C for 30 minutes, 42 C for 30minutes, followed by 85 C for 5 minutes, and then held at 4 C. Thereactions were diluted 4 times by adding 30 ul of dH20 prior to the PCRamplification.

A 10 ul PCR amplification was then set up comprising: 2 ul of dilutedreverse transcription reaction product, 1.3 ul 10 uM miRNA specificForward Primer, 0.7 ul 10 uM Universal Reverse Primer, 0.2 ul TaqMandetector probe, 0.2 ul dNTPs (25 mM each), 0.6 ul dH20, 5 ul 2X TaqManmaster mix (Applied Biosystems, without UNG). The reaction was startedwith a 95 C step for 10 minutes. Then, 40 cycles were performed, eachcycle comprising 95 C for 15 seconds, and 60 C for 1 minute. Table 1indicates the results of this experiment.

TABLE 1 Reverse miRNA Replicate Ligase transcriptase Let-7a1 mir16 mir20mir21 mir26a mir30a mir224 average Yes Yes 16.8 16.0 19.1 16.8 15.0 21.327.3 18.9 Yes No 38.7 31.3 39.9 31.9 30.1 33.3 40.0 35.0 I No Yes 18.014.6 18.3 16.2 14.0 21.3 26.4 18.4 No No 40.0 36.6 40.0 40.0 33.8 39.240.0 38.5 Yes Yes 17.1 16.2 19.3 17.0 15.1 21.4 27.3 19.1 Yes No 38.931.2 37.6 32.1 30.4 33.4 39.4 34.7 II No Yes 18.4 14.8 18.7 16.6 14.321.5 26.7 18.7 No No 40.0 36.1 40.0 40.0 34.1 40.0 40.0 38.6 ReplicateYes Yes 16.9 16.1 19.2 16.9 15.0 21.4 27.3 19.0 Average Yes No 38.8 31.238.8 32.0 30.3 33.4 39.7 34.9 No Yes 18.2 14.7 18.5 16.4 14.1 21.4 26.618.6 No No 40.0 36.4 40.0 40.0 34.0 39.6 40.0 40.0

Sequences of corresponding forward primers, reverse primer, and TaqManprobes are shown in Table 2.

TABLE 2 miRNA ID miRNA sequences miR-16 uagcagcacguaaauauuggcg miR-20uaaagugcuuauagugcaggua miR-21 uagcuuaucagacugauguuga miR-22aagcugccaguugaagaacugu miR-26a uucaaguaauccaggauaggcu miR-29cuagcaccaucugaaaucgguu miR-30a cuuucagucggauguuugcagc miR-34uggcagugucuuagcugguugu miR-200b cucuaauacugccugguaaugaug miR-323gcacauuacacggucgaccucu miR-324-5 cgcauccccuagggcauuggugu let-7a1ugagguaguagguuguauaguu Linker probe Linker probe sequences miR-16linR6GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCGCCAA miR20LinR6GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTACCTG miR-21linR6GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACA miR-22linR6GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGTT miR-26alinR6GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGCCTA miR-29linR6GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACCGA miR30LinR6GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTGCA miR-34linR6GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAACC miR-200blinR6GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCATCAT miR-323linR6GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGAGGT miR-324-5linR6GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACACCA let7aLinR6GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTAT Forward primer IDForward primer sequences miR-16F55 CGCGCTAGCAGCACGTAAAT miR-20F56GCCGCTAAAGTGCTTATAGTGC miR-21F56 GCCCGCTAGCTTATCAGACTGATG miR-22F56GCCTGAAGCTGCCAGTTGA miR-26aF54 CCGGCGTTCAAGTAATCCAGGA miR-29F56GCCGCTAGCACCATCTGAAA miR-30aF58 GCCCCTTTCAGTCGGATGTTT miR-34F56GCCCGTGGCAGTGTCTTAG miR-200bF56 GCCCCTCTAATACTGCCTGG miR-323F58GCCACGCACATTACACGGTC miR-324-5F56 GCCACCATCCCCTAGGGC let-7a1F56GCCGCTGAGGTAGTAGGTTGT TaqMan probe ID TaqMan probe sequencesmiR-16Tq8F67 (6FAM)ATACGACCGCCAATAT(MGB) miR20_Tq8F68(6FAM)CTGGATACGACTACCTG(MGB) miR-21_Tq8F68 (6FAM)CTGGATACGACTCAACA(MGB)miR-22_Tq8F68 (6FAM)TGGATACGACACAGTTCT(MGB) miR-26a_Tq8F69(6FAM)TGGATACGACAGCCTATC(MGB) miR-29_Tq8F68 (6FAM)TGGATACGACAACCGAT(MGB)miR30_Tq8F68 (6FAM)CTGGATACGACGCTGC(MGB) miR-34_Tq8F68(6FAM)ATACGACACAACCAGC(MGB) miR-200b_Tq8F67 (6FAM)ATACGACCATCATTACC(MGB)miR-323_Tq8F67 (6FAM)CTGGATACGACAGAGGT(MGB) miR-324-5Tq8F68(6FAM)ATACGACACACCAATGC(MGB) let7a_Tq8F68 (6FAM)TGGATACGACAACTATAC(MGB)Universal reverse primer ID Reverse primer sequence miR-UP-R67.8GTGCAGGGTCCGAGGT

Example 2

A multiplex (12-plex) assay was performed and the results compared to acorresponding collection of single-plex reactions. Additionally, theeffect of the presence or absence of ligase, as well as the presence orabsence of reverse transcriptase, was determined. The experiments wereperformed essentially the same as in Example 1, and the concentration ofeach linker in the 12-plex reaction was 0.05 uM, thereby resulting in atotal linker probe concentration of 0.6 uM. Further, the diluted 12-plexreverse transcription product was split into 12 different PCRamplification reactions, wherein a miRNA forward primer and a universalreverse primer and a detector probe where in each amplificationreaction. The miRNA sequences, Forward primers, and TaqMan detectorprobes are included in Table 2. The results are shown in Table 3.

TABLE 3 Singleplex vs. Multiplex Assay With Or Without T4 DNA Ligase1-plex Ct 12-plex Ct Ligation + RT 1- vs. 12- miRNA Ligation + RT RTonly Ligation + RT RT only vs RT only plex let-7a1 17.8 16.3 17.6 17.01.0 −0.3 mir-16 16.0 15.1 16.1 15.3 0.9 −0.1 mir-20 19.3 18.7 19.8 19.50.4 −0.6 mir-21 17.0 15.8 17.1 16.3 1.0 −0.3 mir-22 21.6 20.4 21.4 20.71.0 −0.1 mir-26a 15.2 14.3 15.6 14.9 0.8 −0.4 mir-29 17.9 16.8 17.7 17.00.9 0.0 mir-30a 20.7 19.9 21.2 20.7 0.7 −0.7 mir-34 21.3 20.4 22.0 21.00.9 −0.6 mir-200b 19.9 19.2 21.1 20.2 0.8 −1.0 mir-323 32.5 31.2 33.632.3 1.3 −1.1 mir-324-5 24.7 23.1 25.0 24.4 1.1 −0.8 Average 20.3 19.320.7 19.9 0.9 −0.5

Example 3

An experiment was performed to determine the effect of buffer conditionson reaction performance. In one set of experiments, a commerciallyavailable reverse transcription buffer from Applied Biosystems (partnumber 43400550) was employed in the hybridization and extensionreaction. In a corresponding set of experiments, a commerciallyavailable T4 DNA ligase buffer (NEB) was employed in the hybridizationand extension reaction. The experiments were performed as single-plexformat essentially as described for Example 1, and each miRNA was donein triplicate. The results are shown in Table 4, comparing RT buffer (ABpart #4340550) vs T4 DNA ligase buffer.

TABLE 4 RT Buffer T4 DNA Ligase Buffer RT vs I II III Mean I II III MeanT4 Buffer let-7a1 22.7 22.8 22.8 22.8 20.8 20.7 20.6 20.7 2.1 mir-1618.4 18.5 18.6 18.5 17.7 17.8 17.9 17.8 0.7 mir-20 23.6 23.7 23.8 23.723.1 23.1 23.0 23.1 0.6 mir-21 20.4 20.4 20.5 20.4 19.4 19.3 19.2 19.31.1 mir-22 24.0 23.9 24.1 24.0 22.7 22.7 22.7 22.7 1.3 mir-26a 19.8 19.920.1 19.9 18.9 19.0 19.0 18.9 1.0 mir-29 21.3 21.3 21.4 21.3 20.5 20.620.5 20.5 0.8 mir-30a 24.4 24.4 24.4 24.4 23.6 23.4 23.6 23.5 0.9 mir-3424.9 24.8 25.1 25.0 23.0 23.1 23.2 23.1 1.9 mir-200b 25.8 25.8 25.9 25.924.6 24.6 24.8 24.7 1.2 mir-323 34.6 34.5 34.8 34.6 34.7 34.2 34.5 34.50.2 mir-324-5 26.0 26.0 26.1 26.0 25.4 25.7 25.6 25.6 0.5 Average 23.823.8 24.0 23.9 22.9 22.8 22.9 22.9 1.0

Example 4

An experiment was performed to examine the effect of ligase and kinasein a real-time miRNA amplification reaction. Here, twelve single-plexreactions were performed in duplicate, essentially as described inExample 1. Results are shown in Table 5.

TABLE 5 Ligase & Kinase No Ligase/No Kinase I II Mean I II Mean let-7a117.7 17.9 17.8 16.2 16.4 16.3 mir-16 15.9 16.2 16.0 15.0 15.2 15.1mir-20 19.1 19.6 19.3 18.6 18.9 18.7 mir-21 16.9 17.2 17.0 15.7 15.915.8 mir-22 21.4 21.7 21.6 20.3 20.5 20.4 mir-26a 15.0 15.4 15.2 14.314.4 14.3 mir-29 17.9 18.0 17.9 16.7 16.8 16.8 mir-30a 20.6 20.8 20.719.8 20.0 19.9 mir-34 21.1 21.5 21.3 20.4 20.5 20.4 mir-200b 19.8 20.019.9 19.2 19.3 19.2 mir-323 32.3 32.6 32.5 31.1 31.2 31.2 mir-324-5 24.624.8 24.7 23.0 23.3 23.1 Average 20.2 20.5 20.3 19.2 19.4 19.3

Example 5

An experiment was performed to determine the effect of sample materialon Ct values in a real-time miRNA amplification reaction. Here, cells,GuHCl lysate, Tris lysate, and Purified RNA were compared. The cellswere NIH3T3 cells. The Purified RNA was collected using the commerciallyavailable mirVana mRNA isolation kit for Ambion (catalog number 1560). ATris lysate, and a Guanidine lysate (GuHCl) (commercially available fromApplied Biosystems), were prepared as follows:

For the Tris lysate, a 1× lysis buffer comprised 10 mM Tris-HCl, pH 8.0,0.02% Sodium Azide, and 0.03% Tween-20. Trypsinized cells were pelletedby centrifugation at 1500 rpm for 5 minutes. The growth media wasremoved by aspiration, being careful that the cell pellet was notdisturbed. PBS was added to bring the cells to 2×10³ cells/ul. Next 10ul of cell suspension was mixed with 10 ul of a 2× lysis buffer and spunbriefly. The tubes were then immediately incubated for 5 minutes at 95C, and then immediately placed in a chilled block on ice for 2 minutes.The tubes were then mixed well and spun briefly at full speed before use(or optionally, stored at −20 C).

For the GuHCl lysate, a 1× lysis buffer comprised 2.5M GuHCl, 150 mM MESpH 6.0, 200 mM NaCl, 0.75% Tween-20. Trypsinized cells were pelleted bycentrifugation at 1500 rpm for 5 minutes. The growth media was removedby aspiration, being careful that the cell pellet was not disturbed. Thecell pellet was then re-suspended in 1×PBS, Ca++ and Mg++ free to bringcells to 2×10⁴ cells/uL. Then, 1 volume of 2× lysis buffer was added. Toensure complete nucleic acid release, this was followed by pipetting upand down ten times, followed by a brief spin. Results are shown in Table6.

Similar results were obtained for a variety of cell lines, including.NIH/3T3, OP9, A549, and HepG2 cells.

TABLE 6 Ct miRNA ID Cells GuHCl lysate Tris lysate Purified RNA let-7a124.9 31.3 28.2 31.5 mir-16 22.3 25.2 22.3 24.9 mir-20 22.7 26.0 24.126.1 mir-21 21.3 24.2 22.0 24.7 mir-22 30.3 28.6 27.2 28.8 mir-26a 25.631.0 27.9 31.4 mir-29 27.2 27.9 26.5 27.4 mir-30a 26.1 32.2 28.9 30.7mir-34 26.8 30.3 26.4 27.4 mir-200b 40.0 40.0 40.0 40.0 mir-323 30.134.7 31.1 31.8 mir-324-5 28.6 29.7 28.3 29.3 Average 27.2 30.1 27.8 29.5

Example 6

An experiment was performed to demonstrate the ability of the reactionto selectively quantity mature miRNA in the presence of precursor miRNA.Here, let-7a miRNA and mir-26b miRNA were queried in both mature form aswell as in their precursor form. Experiments were performed essentiallyas described for Example 1 in the no ligase condition, done intriplicate, with varying amounts of target material as indicated.Results are shown in Table 7. The sequences examined were as follows:

Mature let-7a, Seq ID NO: UGAGGUAGUAGGUUGUAUAGUUPrecursor let-7a, SEQ ID NO:(Note that the underlined sequences corresponds to the Mature let-7a.)GGGUGAGGUAGUAGGUUGUAUAGUUUGGGGCUCUGCCCUGCUAUGGGAUAACUAUACAAUCUACUGUCUUUCCU Mature mir-26b, SEQ ID NO:UUCAAGUAAUUCAGGAUAGGU Precursor mir-26b of SEQ ID NO:(Note that the underlined sequences corresponds to the Mature mir-26b.)CCGGGACCCAGUUCAAGUAAUUCAGGAUAGGUUGUGUGCUGUCCAGCCUGUUCUCCAUUACUUGGCUCGGGGACCGG

TABLE 7 Mouse Synthetic Synthetic lung miRNA precursor Assay specificfor (CT) Target RNA (ng) (fM) (fM) miRNA Precursor Let-7a 0 0 0 40.0 ±0.0 40.0 ± 0.0 (let-7a3) 0 10 0 24.2 ± 0.3 40.0 ± 0.0 0 100 0 21.0 ± 0.240.0 ± 0.0 0 0 10 35.0 ± 1.0 25.0 ± 0.1 0 0 100 31.0 ± 0.1 21.5 ± 0.1 100 0 19.1 ± 0.4 40.0 ± 0.0 Mir-26b 0 0 0 40.0 ± 0.0 40.0 ± 0.0 0 10 023.1 ± 0.1 40.0 ± 0.0 0 100 0 19.7 ± 0.1 40.0 ± 0.0 0 0 10 32.9 ± 0.425.7 ± 0.0 0 0 100 28.9 ± 0.2 22.3 ± 0.0 10 0 0 20.5 ± 0.1 28.0 ± 0.2

Example 7

An experiment was performed on synthetic let-7a miRNA to assess thenumber of 3′ nucleotides in the 3′ target specific portion of the linkerprobe that correspond with the 3′ end region of the miRNA. Theexperiment was performed as essentially as described supra for Example 1for the no ligase condition, and results are shown in Table 8 as meansand standard deviations of Ct values.

TABLE 8 miRNA assay components: let-7a miRNA synthetic target: let-7aNo. 3′ ssDNA linker probe target C_(T) values & statistics specificportion bases I II III Average SD 7 29.4 29.1 29.3 29.3 0.1 6 30.1 29.930.2 30.1 0.2 5 33.9 33.2 33.8 33.6 0.4 4 40.0 39.2 40.0 39.7 0.4In some embodiments, 3′ target specific portions of linker probespreferably comprise 5 nucleotides that correspond to the 3′ end regionof miRNAs. For example, miR-26a and miR-26b differ by only 2 bases, oneof which is the 3′ end nucleotide of miR-26a. Linker probes comprising 5nucleotides at their 3′ target specific portions can be employed toselectively detect miR-26a versus miR-26b.

Additional strategies for using the linker probes of the presentteachings in the context of single step assays, as well as in thecontext of short primer compositions, can be found in filed U.S.Provisional application “Compositions, Methods, and Kits for Identifyingand Quantitating Small RNA Molecules” by Lao and Straus, as well as inElfaitouri et al., J. Clin. Virol. 2004, 30(2): 150-156.

The present teachings further contemplate linker probe compositionscomprising 3′ target specific portions corresponding to any micro RNAsequence, including but without limitation, those sequences shown inTable 9, including C. elegans (cel), mouse (mmu), human (hsa),drosophila (dme), rat (mo), and rice (osa).

TABLE 9 cel-let-7 mmu-let-7g ugagguaguagguuguauaguuugagguaguaguuuguacagu cel-lin-4 mmu-let-7i ucccugagaccucaagugugaugagguaguaguuugugcu cel-miR-1 mmu-miR-1 uggaauguaaagaaguauguauggaauguaaagaaguaugua cel-miR-2 mmu-miR-15b uaucacagccagcuuugaugugcuagcagcacaucaugguuuaca cel-miR-34 mmu-miR-23b aggcagugugguuagcugguugaucacauugccagggauuaccac cel-miR-35 mmu-miR-27b ucaccggguggaaacuagcaguuucacaguggcuaaguucug cel-miR-36 mmu-miR-29b ucaccgggugaaaauucgcauguagcaccauuugaaaucagugu cel-miR-37 mmu-miR-30a* ucaccgggugaacacuugcaguuguaaacauccucgacuggaagc cel-miR-38 mmu-miR-30a ucaccgggagaaaaacuggagucuuucagucggauguuugcagc cel-miR-39 mmu-miR-30b ucaccggguguaaaucagcuuguguaaacauccuacacucagc cel-miR-40 mmu-miR-99a ucaccggguguacaucagcuaaacccguagauccgaucuugu cel-miR-41 mmu-miR-99b ucaccgggugaaaaaucaccuacacccguagaaccgaccuugcg cel-miR-42 mmu-miR-101 caccggguuaacaucuacaguacaguacugugauaacuga cel-miR-43 mmu-miR-124a uaucacaguuuacuugcugucgcuuaaggcacgcggugaaugcca cel-miR-44 mmu-miR-125a ugacuagagacacauucagcuucccugagacccuuuaaccugug cel-miR-45 mmu-miR-125b ugacuagagacacauucagcuucccugagacccuaacuuguga cel-miR-46 mmu-miR-126* ugucauggagucgcucucuucacauuauuacuuuugguacgcg cel-miR-47 mmu-miR-126 ugucauggaggcgcucucuucaucguaccgugaguaauaaugc cel-miR-48 mmu-miR-127 ugagguaggcucaguagaugcgaucggauccgucugagcuuggcu cel-miR-49 mmu-miR-128a aagcaccacgagaagcugcagaucacagugaaccggucucuuuu cel-miR-50 mmu-miR-130a ugauaugucugguauucuuggguucagugcaauguuaaaagggc cel-miR-51 mmu-miR-9 uacccguagcuccuauccauguuucuuugguuaucuagcuguauga cel-miR-52 mmu-miR-9* cacccguacauauguuuccgugcuuaaagcuagauaaccgaaagu cel-miR-53 mmu-miR-132 cacccguacauuuguuuccgugcuuaacagucuacagccauggucg cel-miR-54 mmu-miR-133a uacccguaaucuucauaauccgaguugguccccuucaaccagcugu cel-miR-55 mmu-miR-134 uacccguauaaguuucugcugagugugacugguugaccagaggg cel-miR-56* mmu-miR-135a uggcggauccauuuuggguuguauauggcuuuuuauuccuauguga cel-miR-56 mmu-miR-136 uacccguaauguuuccgcugagacuccauuuguuuugaugaugga cel-miR-57 mmu-miR-137 uacccuguagaucgagcuguguguuauugcuuaagaauacgcguag cel-miR-58 mmu-miR-138 ugagaucguucaguacggcaauagcugguguugugaauc cel-miR-59 mmu-miR-140 ucgaaucguuuaucaggaugaugagugguuuuacccuaugguag cel-miR-60 mmu-miR-141 uauuaugcacauuuucuaguucaaacacugucugguaaagaugg cel-miR-61 mmu-miR-142-5p ugacuagaaccguuacucaucuccauaaaguagaaagcacuac cel-miR-62 mmu-miR-142-3p ugauauguaaucuagcuuacaguguaguguuuccuacuuuaugg cel-miR-63 mmu-miR-144 uaugacacugaagcgaguuggaaauacaguauagaugauguacuag cel-miR-64 mmu-miR-145 uaugacacugaagcguuaccgaaguccaguuuucccaggaaucccuu cel-miR-65 mmu-miR-146 uaugacacugaagcguaaccgaaugagaacugaauuccauggguu cel-miR-66 mmu-miR-149 caugacacugauuagggaugugaucuggcuccgugucuucacucc cel-miR-67 mmu-miR-150 ucacaaccuccuagaaagaguagaucucccaacccuuguaccagug cel-miR-68 mmu-miR-151 ucgaagacucaaaaguguagacuagacugaggcuccuugagg cel-miR-69 mmu-miR-152 ucgaaaauuaaaaaguguagaucagugcaugacagaacuugg cel-miR-70 mmu-miR-153 uaauacgucguugguguuuccauuugcauagucacaaaaguga cel-miR-71 mmu-miR-154 ugaaagacauggguagugauagguuauccguguugccuucg cel-miR-72 mmu-miR-155 aggcaagauguuggcauagcuuaaugcuaauugugauagggg cel-miR-73 mmu-miR-10b uggcaagauguaggcaguucagucccuguagaaccgaauuugugu cel-miR-74 mmu-miR-129 uggcaagaaauggcagucuacacuuuuugcggucugggcuugcu cel-miR-75 mmu-miR-181a uuaaagcuaccaaccggcuucaaacauucaacgcugucggugagu cel-miR-76 mmu-miR-182 uucguuguugaugaagccuugauuuggcaaugguagaacucaca cel-miR-77 mmu-miR-183 uucaucaggccauagcuguccauauggcacugguagaauucacug cel-miR-78 mmu-miR-184 uggaggccugguuguuugugcuggacggagaacugauaagggu cel-miR-79 mmu-miR-185 auaaagcuagguuaccaaagcuuggagagaaaggcaguuc cel-miR-227 mmu-miR-186 agcuuucgacaugauucugaaccaaagaauucuccuuuugggcuu cel-miR-80 mmu-miR-187 ugagaucauuaguugaaagccgaucgugucuuguguugcagccgg cel-miR-81 mmu-miR-188 ugagaucaucgugaaagcuagucaucccuugcaugguggagggu cel-miR-82 mmu-miR-189 ugagaucaucgugaaagccagugugccuacugagcugauaucagu cel-miR-83 mmu-miR-24 uagcaccauauaaauucaguaauggcucaguucagcaggaacag cel-miR-84 mmu-miR-190 ugagguaguauguaauauuguaugauauguuugauauauuaggu cel-miR-85 mmu-miR-191 uacaaaguauuugaaaagucgugccaacggaaucccaaaagcagcu cel-miR-86 mmu-miR-193 uaagugaaugcuuugccacagucaacuggccuacaaagucccag cel-miR-87 mmu-miR-194 gugagcaaaguuucagguguuguaacagcaacuccaugugga cel-miR-90 mmu-miR-195 ugauauguuguuugaaugccccuagcagcacagaaauauuggc cel-miR-124 mmu-miR-199a uaaggcacgcggugaaugccacccaguguucagacuaccuguuc cel-miR-228 mmu-miR-199a*aauggcacugcaugaauucacgg uacaguagucugcacauugguu cel-miR-229 mmu-miR-200baaugacacugguuaucuuuuccaucgu uaauacugccugguaaugaugac cel-miR-230mmu-miR-201 guauuaguugugcgaccaggaga uacucaguaaggcauuguucu cel-miR-231mmu-miR-202 uaagcucgugaucaacaggcagaa agagguauagcgcaugggaaga cel-miR-232mmu-miR-203 uaaaugcaucuuaacugcgguga ugaaauguuuaggaccacuag cel-miR-233mmu-miR-204 uugagcaaugcgcaugugcggga uucccuuugucauccuaugccug cel-miR-234mmu-miR-205 uuauugcucgagaauacccuu uccuucauuccaccggagucug cel-miR-235mmu-miR-206 uauugcacucuccccggccuga uggaauguaaggaagugugugg cel-miR-236mmu-miR-207 uaauacugucagguaaugacgcu gcuucuccuggcucuccucccuc cel-miR-237mmu-miR-122a ucccugagaauucucgaacagcuu uggagugugacaaugguguuugucel-miR-238 mmu-miR-143 uuuguacuccgaugccauucaga ugagaugaagcacuguagcucacel-miR-239a mmu-miR-30e uuuguacuacacauagguacugg uguaaacauccuugacuggacel-miR-239b mmu-miR-290 uuguacuacacaaaaguacug cucaaacuaugggggcacuuuuucel-miR-240 mmu-miR-291-5p uacuggcccccaaaucuucgcu caucaaaguggaggcccucucucel-miR-241 mmu-miR-291-3p ugagguaggugcgagaaauga aaagugcuuccacuuugugugcccel-miR-242 mmu-miR-292-5p uugcguaggccuuugcuucga acucaaacugggggcucuuuugcel-miR-243 mmu-miR-292-3p cgguacgaucgcggcgggauaucaagugccgccagguuuugagugu cel-miR-244 mmu-miR-293 ucuuugguuguacaaagugguaugagugccgcagaguuuguagugu cel-miR-245 mmu-miR-294 auugguccccuccaaguagcucaaagugcuucccuuuugugugu cel-miR-246 mmu-miR-295 uuacauguuucggguaggagcuaaagugcuacuacuuuugagucu cel-miR-247 mmu-miR-296 ugacuagagccuauucucuucuuagggcccccccucaauccugu cel-miR-248 mmu-miR-297 uacacgugcacggauaacgcucaauguaugugugcaugugcaug cel-miR-249 mmu-miR-298 ucacaggacuuuugagcguugcggcagaggagggcuguucuucc cel-miR-250 mmu-miR-299 ucacagucaacuguuggcauggugguuuaccgucccacauacau cel-miR-251 mmu-miR-300 uuaaguaguggugccgcucuuauuuaugcaagggcaagcucucuuc cel-miR-252 mmu-miR-301 uaaguaguagugccgcagguaaccagugcaauaguauugucaaagc cel-miR-253 mmu-miR-302 cacaccucacuaacacugaccuaagugcuuccauguuuugguga cel-miR-254 mmu-miR-34c ugcaaaucuuucgcgacuguaggaggcaguguaguuagcugauugc cel-miR-256 mmu-miR-34b uggaaugcauagaagacuguauaggcaguguaauuagcugauug cel-miR-257 mmu-let-7d gaguaucaggaguacccagugaagagguaguagguugcauagu cel-miR-258 mmu-let-7d* gguuuugagaggaauccuuuucuauacgaccugcugccuuucu cel-miR-259 mmu-miR-106a aaaucucauccuaaucugguacaaagugcuaacagugcaggua cel-miR-260 mmu-miR-106b gugaugucgaacucuuguaguaaagugcugacagugcagau cel-miR-261 mmu-miR-130b uagcuuuuuaguuuucacgcagugcaaugaugaaagggcau cel-miR-262 mmu-miR-19b guuucucgauguuuucugauugugcaaauccaugcaaaacuga cel-miR-264 mmu-miR-30c ggcgggugguuguuguuauguguaaacauccuacacucucagc cel-miR-265 mmu-miR-30d ugagggaggaagggugguauuguaaacauccccgacuggaag cel-miR-266 mmu-miR-148a 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dme-miR-2buccuucauuccaccggagucug uaucacagccagcuuugaggagc hsa-miR-210 dme-miR-3cugugcgugugacagcggcug ucacugggcaaagugugucuca hsa-miR-211 dme-miR-4uucccuuugucauccuucgccu auaaagcuagacaaccauuga hsa-miR-212 dme-miR-5uaacagucuccagucacggcc aaaggaacgaucguugugauaug hsa-miR-213 dme-miR-6accaucgaccguugauuguacc uaucacaguggcuguucuuuuu hsa-miR-214 dme-miR-7acagcaggcacagacaggcag uggaagacuagugauuuuguugu hsa-miR-215 dme-miR-8augaccuaugaauugacagac uaauacugucagguaaagauguc hsa-miR-216 dme-miR-9auaaucucagcuggcaacugug ucuuugguuaucuagcuguauga hsa-miR-217 dme-miR-10uacugcaucaggaacugauuggau acccuguagauccgaauuugu hsa-miR-218 dme-miR-11uugugcuugaucuaaccaugu caucacagucugaguucuugc hsa-miR-219 dme-miR-12ugauuguccaaacgcaauucu ugaguauuacaucagguacuggu hsa-miR-220 dme-miR-13accacaccguaucugacacuuu uaucacagccauuuugaugagu hsa-miR-221 dme-miR-13bagcuacauugucugcuggguuuc uaucacagccauuuugacgagu hsa-miR-222 dme-miR-14agcuacaucuggcuacugggucuc ucagucuuuuucucucuccua hsa-miR-223 dme-miR-263augucaguuugucaaauacccc 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agagguaguagguugcauagu hsa-miR-342rno-let-7d* ucucacacagaaaucgcacccguc cuauacgaccugcugccuuucu hsa-miR-337rno-miR-328 uccagcuccuauaugaugccuuu cuggcccucucugcccuuccgu hsa-miR-323rno-miR-329 gcacauuacacggucgaccucu aacacacccagcuaaccuuuuu hsa-miR-326rno-miR-330 ccucugggcccuuccuccag gcaaagcacagggccugcagaga hsa-miR-151rno-miR-331 acuagacugaagcuccuugagg gccccugggccuauccuagaa hsa-miR-135brno-miR-333 uauggcuuuucauuccuaugug guggugugcuaguuacuuuu hsa-miR-148brno-miR-140 ucagugcaucacagaacuuugu agugguuuuacccuaugguag hsa-miR-331rno-miR-140* gccccugggccuauccuagaa uaccacaggguagaaccacggacahsa-miR-324-5p rno-miR-336 cgcauccccuagggcauuggugu ucacccuuccauaucuagucuhsa-miR-324-3p rno-miR-337 ccacugccccaggugcugcugguucagcuccuauaugaugccuuu hsa-miR-338 mo-miR-148b uccagcaucagugauuuuguugaucagugcaucacagaacuuugu hsa-miR-339 rno-miR-338 ucccuguccuccaggagcucauccagcaucagugauuuuguuga hsa-miR-335 rno-miR-339 ucaagagcaauaacgaaaaauguucccuguccuccaggagcuca hsa-miR-133b rno-miR-341 uugguccccuucaaccagcuaucgaucggucggucggucagu rno-miR-342 ucucacacagaaaucgcacccguc osa-miR156rno-miR-344 ugacagaagagagugagcac ugaucuagccaaagccugaccgu osa-miR160rno-miR-345 ugccuggcucccuguaugcca ugcugaccccuaguccagugc osa-miR162rno-miR-346 ucgauaaaccucugcauccag ugucugccugagugccugccucu osa-miR164rno-miR-349 uggagaagcagggcacgugca cagcccugcugucuuaaccucu osa-miR166rno-miR-129 ucggaccaggcuucauucccc cuuuuugcggucugggcuugcu osa-miR167rno-miR-129* ugaagcugccagcaugaucua aagcccuuaccccaaaaagcau osa-miR169rno-miR-20 cagccaaggaugacuugccga uaaagugcuuauagugcagguag osa-miR171rno-miR-20* ugauugagccgcgccaauauc acugcauuacgagcacuuaca rno-miR-350uucacaaagcccauacacuuucac rno-miR-7 uggaagacuagugauuuuguu rno-miR-7*caacaaaucacagucugccaua rno-miR-351 ucccugaggagcccuuugagccug rno-miR-135buauggcuuuucauuccuaugug rno-miR-151* ucgaggagcucacagucuagua rno-miR-151acuagacugaggcuccuugagg rno-miR-101b uacaguacugugauagcugaag rno-let-7augagguaguagguuguauaguu rno-let-7b ugagguaguagguugugugguu rno-let-7cugagguaguagguuguaugguu rno-let-7e ugagguaggagguuguauagu rno-let-7fugagguaguagauuguauaguu rno-let-7i ugagguaguaguuugugcu rno-miR-7buggaagacuugugauuuuguu rno-miR-9 ucuuugguuaucuagcuguauga rno-miR-10auacccuguagauccgaauuugug rno-miR-10b uacccuguagaaccgaauuugu rno-miR-15buagcagcacaucaugguuuaca rno-miR-16 uagcagcacguaaauauuggcg rno-miR-17caaagugcuuacagugcagguagu rno-miR-18 uaaggugcaucuagugcagaua rno-miR-19bugugcaaauccaugcaaaacuga rno-miR-19a ugugcaaaucuaugcaaaacuga rno-miR-21uagcuuaucagacugauguuga rno-miR-22 aagcugccaguugaagaacugu rno-miR-23aaucacauugccagggauuucc rno-miR-23b aucacauugccagggauuaccac rno-miR-24uggcucaguucagcaggaacag rno-miR-25 cauugcacuugucucggucuga rno-miR-26auucaaguaauccaggauaggcu rno-miR-26b uucaaguaauucaggauagguu rno-miR-27buucacaguggcuaaguucug rno-miR-27a uucacaguggcuaaguuccgc rno-miR-28aaggagcucacagucuauugag rno-miR-29b uagcaccauuugaaaucagugu rno-miR-29acuagcaccaucugaaaucgguu rno-miR-29c uagcaccauuugaaaucgguua rno-miR-30cuguaaacauccuacacucucagc rno-miR-30e uguaaacauccuugacugga rno-miR-30buguaaacauccuacacucagc rno-miR-30d uguaaacauccccgacuggaag rno-miR-30acuuucagucggauguuugcagc rno-miR-31 aggcaagaugcuggcauagcug rno-miR-32uauugcacauuacuaaguugc rno-miR-33 gugcauuguaguugcauug rno-miR-34buaggcaguguaauuagcugauug rno-miR-34c aggcaguguaguuagcugauugc rno-miR-34auggcagugucuuagcugguuguu rno-miR-92 uauugcacuugucccggccug rno-miR-93caaagugcuguucgugcagguag rno-miR-96 uuuggcacuagcacauuuuugcu rno-miR-98ugagguaguaaguuguauuguu rno-miR-99a aacccguagauccgaucuugug rno-miR-99bcacccguagaaccgaccuugcg rno-miR-100 aacccguagauccgaacuugug rno-miR-101uacaguacugugauaacugaag rno-miR-103 agcagcauuguacagggcuauga rno-miR-106buaaagugcugacagugcagau rno-miR-107 agcagcauuguacagggcuauca rno-miR-122auggagugugacaaugguguuugu rno-miR-124a uuaaggcacgcggugaaugcca rno-miR-125aucccugagacccuuuaaccugug rno-miR-125b ucccugagacccuaacuuguga rno-miR-126*cauuauuacuuuugguacgcg rno-miR-126 ucguaccgugaguaauaaugc rno-miR-127ucggauccgucugagcuuggcu rno-miR-128a ucacagugaaccggucucuuuu rno-miR-128bucacagugaaccggucucuuuc rno-miR-130a cagugcaauguuaaaagggc rno-miR-130bcagugcaaugaugaaagggcau rno-miR-132 uaacagucuacagccauggucg rno-miR-133auugguccccuucaaccagcugu rno-miR-134 ugugacugguugaccagaggg rno-miR-135auauggcuuuuuauuccuauguga rno-miR-136 acuccauuuguuuugaugaugga rno-miR-137uauugcuuaagaauacgcguag rno-miR-138 agcugguguugugaauc rno-miR-139ucuacagugcacgugucu rno-miR-141 aacacugucugguaaagaugg rno-miR-142-5pcauaaaguagaaagcacuac rno-miR-142-3p uguaguguuuccuacuuuaugga rno-miR-143ugagaugaagcacuguagcuca rno-miR-144 uacaguauagaugauguacuag rno-miR-145guccaguuuucccaggaaucccuu rno-miR-146 ugagaacugaauuccauggguu rno-miR-150ucucccaacccuuguaccagug rno-miR-152 ucagugcaugacagaacuugg rno-miR-153uugcauagucacaaaaguga rno-miR-154 uagguuauccguguugccuucg rno-miR-181caacauucaaccugucggugagu rno-miR-181a aacauucaacgcugucggugagu rno-miR-181baacauucauugcugucgguggguu rno-miR-183 uauggcacugguagaauucacug rno-miR-184uggacggagaacugauaagggu rno-miR-185 uggagagaaaggcaguuc rno-miR-186caaagaauucuccuuuugggcuu rno-miR-187 ucgugucuuguguugcagccg rno-miR-190ugauauguuugauauauuaggu rno-miR-191 caacggaaucccaaaagcagcu rno-miR-192cugaccuaugaauugacagcc rno-miR-193 aacuggccuacaaagucccag rno-miR-194uguaacagcaacuccaugugga rno-miR-195 uagcagcacagaaauauuggc rno-miR-196uagguaguuucauguuguugg rno-miR-199a cccaguguucagacuaccuguuc rno-miR-200caauacugccggguaaugaugga rno-miR-200a uaacacugucugguaacgaugu rno-miR-200bcucuaauacugccugguaaugaug rno-miR-203 gugaaauguuuaggaccacuag rno-miR-204uucccuuugucauccuaugccu rno-miR-205 uccuucauuccaccggagucug rno-miR-206uggaauguaaggaagugugugg rno-miR-208 auaagacgagcaaaaagcuugu rno-miR-210cugugcgugugacagcggcug rno-miR-211 uucccuuugucauccuuugccu rno-miR-212uaacagucuccagucacggcc rno-miR-213 accaucgaccguugauuguacc rno-miR-214acagcaggcacagacaggcag rno-miR-216 uaaucucagcuggcaacugug rno-miR-217uacugcaucaggaacugacuggau rno-miR-218 uugugcuugaucuaaccaugu rno-miR-219ugauuguccaaacgcaauucu rno-miR-221 agcuacauugucugcuggguuuc rno-miR-222agcuacaucuggcuacugggucuc rno-miR-223 ugucaguuugucaaauacccc rno-miR-290cucaaacuaugggggcacuuuuu rno-miR-291-5p caucaaaguggaggcccucucurno-miR-291-3p aaagugcuuccacuuugugugcc rno-miR-292-5pacucaaacugggggcucuuuug rno-miR-292-3p aagugccgccagguuuugagugurno-miR-296 agggcccccccucaauccugu rno-miR-297 auguaugugugcauguaugcaugrno-miR-298 ggcagaggagggcuguucuucc rno-miR-299 ugguuuaccgucccacauacaurno-miR-300 uaugcaagggcaagcucucuuc rno-miR-320 aaaagcuggguugagagggcgaarno-miR-321 uaagccagggauuguggguuc

Although the disclosed teachings have been described with reference tovarious applications, methods, kits, and compositions, it will beappreciated that various changes and modifications may be made withoutdeparting from the teachings herein. The foregoing examples are providedto better illustrate the disclosed teachings and are not intended tolimit the scope of the teachings herein.

1. A method for detecting a micro RNA (miRNA) comprising; hybridizingthe miRNA and a linker probe, wherein the linker probe comprises a stem,a loop, and a 3′ target-specific portion, wherein the 3′ target-specificportion base pairs with the 3′ end region of the miRNA; extending thelinker probe to form an extension reaction product; amplifying theextension reaction product to form an amplification product; and,detecting the miRNA.
 2. The method according to claim 1 wherein theamplification reaction is a polymerase chain reaction, wherein theamplification reaction comprises a forward primer that corresponds tothe miRNA, and a reverse primer that corresponds to the linker probe. 3.The method according to claim 1 wherein the miRNA is 18-25ribonucleotides in length.
 4. The method according to claim 1 whereinthe amplification reaction comprises a detector probe.
 5. The methodaccording to claim 4 wherein the detector probe comprises a nucleotideof the linker probe in the amplification product or a nucleotide of thelinker probe complement in the amplification product.
 6. The methodaccording to claim 4 wherein the detector probe comprises a nucleotideof the linker probe stem in the amplification product or a nucleotide ofthe linker probe stem complement in the amplification product.
 7. Themethod according to claim 4 wherein the detector probe comprises anucleotide of the 3′ end region of the miRNA in the amplificationproduct or a nucleotide of the 3′ end region of the miRNA complement inthe amplification product.
 8. The method according to claim 4 whereinthe detector probe comprises a nucleotide of a region upstream from the3′ end region of the miRNA in the amplification product or a nucleotideof a region upstream from the 3′ end region of the miRNA complement inthe amplification product.
 9. The method according to claim 4 whereinthe detector probe is a 5′-nuclease cleavable probe.
 10. The methodaccording to claim 9 wherein the 5′-nuclease cleavable probe comprisesFAM.
 11. The method according to claim 9 wherein the 5′-nucleasecleavable probe comprises VIC.
 12. The method according to claim 4wherein the detector probe comprises peptide nucleic acid (PNA).
 13. Themethod according to claim 12 wherein the PNA probe comprises FAM. 14.The method according to claim 12 wherein the PNA probe comprises VIC.15. The method according to claim 4 wherein the detector probe compriseslocked nucleic acid (LNA).
 16. The method according to claim 4 whereinthe detector probe comprises a universal base.
 17. The method accordingto claim 4 wherein the detector probe is an intercalating dye.
 18. Themethod according to claim 1 wherein the extending is a reversetranscription reaction comprising a reverse transcriptase.
 19. Themethod according to claim 1 wherein the stem of the linker probecomprises 12-16 base-pairs.
 20. The method according to claim 19 whereinthe stem of the linker probe comprises 14 base-pairs.
 21. The methodaccording to claim 1 wherein the 3′ target specific portion of thelinker probe comprises 5-8 nucleotides.
 22. The method according toclaim 1 wherein the loop corresponds to a universal reverse primerportion.
 23. The method according to claim 1 wherein the loop comprises14-18 nucleotides.
 24. The method according to claim 23 wherein the loopcomprises 16 nucleotides.
 25. The method according to claim 4 whereinthe Tm of the detector probe is 63-69 C.
 26. A method for detecting atarget polynucleotide comprising; hybridizing the target polynucleotideand a linker probe, wherein the linker probe comprises a stem, a loop,and a 3′ target-specific portion, wherein the 3′ target-specific portionbase pairs with the 3′ end region of the target polynucleotide;extending the linker probe to form an extension reaction product;amplifying the extension reaction product to form an amplificationproduct in the presence of a detector probe, wherein the detector probecomprises a nucleotide of the linker probe stem in the amplificationproduct or a nucleotide of the linker probe stem complement in theamplification product; and, detecting the target polynucleotide.
 27. Themethod according to claim 26 wherein the amplification reaction is apolymerase chain reaction, wherein the amplification reaction comprisesa forward primer that corresponds to the target polynucleotide, and areverse primer that corresponds to the linker probe.
 28. The methodaccording to claim 26 wherein the target polynucleotide is a micro RNA(miRNA).
 29. The method according to claim 26 wherein the detector probecomprises a nucleotide of the 3′ end region of the target polynucleotidein the amplification product or a nucleotide of the 3′ end region of thetarget polynucleotide complement in the amplification product.
 30. Themethod according to claim 26 wherein the detector probe comprises anucleotide of a region upstream from the 3′ end region of the targetpolynucleotide in the amplification product or a nucleotide of a regionupstream from the 3′ end region of the target polynucleotide complementin the amplification product.
 31. The method according to claim 26wherein the detector probe is a 5′-nuclease cleavable probe.
 32. Themethod according to claim 31 wherein the 5′-nuclease cleavable probecomprises FAM.
 33. The method according to claim 31 wherein the5′-nuclease cleavable probe comprises VIC.
 34. The method according toclaim 26 wherein the detector probe comprises peptide nucleic acid(PNA).
 35. The method according to claim 34 wherein the PNA probecomprises FAM.
 36. The method according to claim 34 wherein the PNAprobe comprises VIC.
 37. The method according to claim 26 wherein thedetector probe comprises locked nucleic acid (LNA).
 38. The methodaccording to claim 26 wherein the detector probe comprises a universalbase.
 39. The method according to claim 26 the extending is a reversetranscription reaction comprising a reverse transcriptase.
 40. Themethod according to claim 26 wherein the stem of the linker probecomprises 12-16 base-pairs.
 41. The method according to claim 40 whereinthe stem of the linker probe comprises 14 base-pairs.
 42. The methodaccording to claim 26 wherein the 3′ target specific portion of thelinker probe comprises 5-8 nucleotides.
 43. The method according toclaim 26 wherein the loop further comprises a universal reverse primerportion.
 44. The method according to claim 26 wherein the loop comprises14-18 nucleotides.
 45. The method according to claim 44 wherein the loopcomprises 16 nucleotides.
 46. The method according to claim 26 whereinthe Tm of the detector probe is 63-69 C.
 47. A method for detecting amiRNA molecule comprising; hybridizing the miRNA molecule and a linkerprobe, wherein the linker probe comprises a stem, a loop, and a 3′target specific portion, wherein the 3′ target-specific portion basepairs with the 3′ end region of the target polynucleotide; extending thelinker probe to form an extension reaction product; amplifying theextension reaction product in the presence of a detector probe to forman amplification product, wherein the detector probe comprises anucleotide of the linker probe stem in the amplification product or anucleotide of the linker probe stem complement in the amplificationproduct, and the detector probe further comprises a nucleotide of the 3′end region of the miRNA in the amplification product or a nucleotide ofthe 3′ end region of the miRNA complement in the amplification product;and, detecting the miRNA molecule.
 48. The method according to claim 47wherein the amplification reaction is a polymerase chain reaction,wherein the amplification reaction comprises a forward primer thatcorresponds to the miRNA, and a reverse primer that corresponds to thelinker probe.
 49. The method according to claim 47 wherein the miRNA is18-25 ribonucleotides in length.
 50. The method according to claim 47wherein the detector probe is a 5′-nuclease cleavable probe.
 51. Themethod according to claim 50 wherein the 5′-nuclease cleavable probecomprises FAM.
 52. The method according to claim 50 wherein the5′-nuclease cleavable probe comprises VIC.
 53. The method according toclaim 47 wherein the detector probe comprises peptide nucleic acid(PNA).
 54. The method according to claim 53 wherein the PNA probecomprises FAM.
 55. The method according to claim 53 wherein the PNAprobe comprises VIC.
 56. The method according to claim 47 wherein thedetector probe comprises locked nucleic acid (LNA).
 57. The methodaccording to claim 47 wherein the detector probe comprises a universalbase.
 58. The method according to claim 47 wherein the extending is areverse transcription reaction comprising a reverse transcriptase. 59.The method according to claim 47 wherein the stem of the linker probecomprises 12-16 base-pairs.
 60. The method according to claim 59 whereinthe stem of the linker probe comprises 14 base-pairs.
 61. The methodaccording to claim 47 wherein the 3′ target specific portion of thelinker probe comprises 5-8 nucleotides.
 62. The method according toclaim 47 wherein the loop further comprises a universal reverse primerportion.
 63. The method according to claim 47 wherein the loop comprises14-18 nucleotides.
 64. The method according to claim 63 wherein the loopcomprises 16 nucleotides.
 65. The method according to claim 47 whereinthe Tm of the detector probe is 63-69 C.
 66. A method for detecting twodifferent miRNAs from a single hybridization reaction comprising;hybridizing a first miRNA and a first linker probe, and a second miRNAand a second linker probe, wherein the first linker probe and the secondlinker probe each comprise a loop, a stem, and a 3′ target-specificportion, wherein the 3′ target-specific portion of the first linkerprobe base pairs with the 3′ end region of the first miRNA, and whereinthe 3′ target-specific portion of the second linker probe base pairswith the 3′ end region of the second miRNA; extending the first linkerprobe and the second linker probe to form extension reaction products;dividing the extension reaction products into a first amplificationreaction to form a first amplification reaction product, and a secondamplification reaction to form a second amplification reaction product,wherein a primer in the first amplification reaction corresponds withthe first miRNA and not the second miRNA, and a primer in the secondamplification reaction corresponds with the second miRNA and not thefirst miRNA, wherein a first detector probe in the first amplificationreaction differs from a second detector probe in the secondamplification reaction, wherein the first detector probe comprises anucleotide of the first linker probe stem of the amplification productor a nucleotide of the first linker probe stem complement in the firstamplification product, wherein the second detector probe comprises anucleotide of the second linker probe stem of the amplification productor a nucleotide of the second linker probe stem complement in theamplification product; and, detecting the two different miRNAs.
 67. Themethod according to claim 66 wherein the first amplification reaction isa first polymerase chain reaction and the second amplification reactionis a second polymerase chain reaction; wherein the first polymerasechain reaction comprises a forward primer that corresponds to the firstmiRNA, and a reverse primer that corresponds to the linker probe,wherein the second polymerase chain reaction comprises a forward primerthat corresponds to the second miRNA, and a reverse primer thatcorresponds to the linker probe, wherein the reverse primer in the firstpolymerase chain reaction and the reverse primer in the secondpolymerase chain reaction are a universal reverse primer.
 68. The methodaccording to claim 66 wherein the first miRNA and/or the second miRNA is18-25 ribonucleotides in length.
 69. The method according to claim 66wherein the first detector probe and/or the second detector probe is a5′-nuclease cleavable probe.
 70. The method according to claim 69wherein the first detector probe and/or the second detector probecomprises FAM.
 71. The method according to claim 69 wherein the firstdetector probe and/or the second detector probe comprises VIC.
 72. Themethod according to claim 66 wherein the first detector probe and/or thesecond detector probe comprises peptide nucleic acid (PNA).
 73. Themethod according to claim 72 wherein first detector probe and/or thesecond detector probe comprises FAM.
 74. The method according to claim72 wherein the first detector probe and/or the second detector probecomprises VIC.
 75. The method according to claim 66 wherein the firstdetector probe and/or the second detector probe comprises locked nucleicacid (LNA).
 76. The method according to claim 66 wherein the firstdetector probe and/or the second detector probe comprises a universalbase.
 77. The method according to claim 66 wherein the extending is areverse transcription reaction comprising a reverse transcriptase. 78.The method according to claim 66 wherein the stem of the first linkerprobe and/or the second linker probe comprises 12-16 base-pairs.
 79. Themethod according to claim 78 wherein the stem of the first linker probeand/or the second linker probe comprises 14 base-pairs.
 80. The methodaccording to claim 66 wherein the 3′ target specific portion of thefirst linker probe and/or the second linker probe comprises 5-8nucleotides.
 81. The method according to claim 66 wherein the loop ofthe first linker probe and/or the second linker probe further comprisesa universal reverse primer portion.
 82. The method according to claim 66wherein the loop of the first linker probe and/or the second linkerprobe comprises 14-18 nucleotides.
 83. The method according to claim 82wherein the loop of the first linker probe and/or the second linkerprobe comprises 16 nucleotides.
 84. The method according to claim 66wherein the Tm of the first detector probe and/or the second detectorprobe is 63-69 C.
 85. A method for detecting two different targetpolynucleotides from a single hybridization reaction comprising;hybridizing a first target polynucleotide and a first linker probe, anda second target polynucleotide and a second linker probe, wherein thefirst linker probe and the second linker probe each comprise a loop, astem, and a 3′ target-specific portion, wherein the 3′ target-specificportion of the first linker probe base pairs with the 3′ end region ofthe first target polynucleotide, and wherein the 3′ target-specificportion of the second linker probe base pairs with the 3′ end region ofthe second target polynucleotide; extending the first linker probe andthe second linker probe to form extension reaction products; dividingthe extension reaction products into a first amplification reaction toform a first amplification reaction product and a second amplificationreaction to form a second amplification reaction product; and, detectingthe two different miRNA molecules.
 86. The method according to claim 85wherein the first amplification reaction is a first polymerase chainreaction and the second amplification reaction is a second polymerasechain reaction; wherein the first polymerase chain reaction comprises aforward primer that corresponds to the first target polynucleotide, anda reverse primer that corresponds to the linker probe, wherein thesecond polymerase chain reaction comprises a forward primer thatcorresponds to the second target polynucleotide, and a reverse primerthat corresponds to the linker probe, wherein the reverse primer in thefirst polymerase chain reaction and the reverse primer in the secondpolymerase chain reaction are a universal reverse primer.
 87. The methodaccording to claim 85 wherein the target polynucleotide is a micro RNA(miRNA).
 88. The method according to claim 85 wherein the firstamplification reaction comprises a first detector probe and/or thesecond amplification reaction comprises a second detector probe.
 89. Themethod according to claim 88 wherein the first detector probecorresponds with a nucleotide of the first linker probe in the firstamplification product or a nucleotide of the first linker probecomplement in the first amplification product, and/or the seconddetector probe corresponds with a nucleotide of the second linker probein the second amplification product or a nucleotide of the second linkerprobe complement in the second amplification product
 90. The methodaccording to claim 88 wherein the first detector probe comprises anucleotide of the first linker probe stem of the first amplificationproduct or a nucleotide of the first linker probe stem complement in thefirst amplification product, and/or the second detector probe comprisesa nucleotide of the second linker probe stem in the second amplificationproduct or a nucleotide of the second linker probe stem complement inthe second amplification product.
 91. The method according to claim 88wherein the first detector probe comprises a nucleotide of the 3′ endregion of the first target polynucleotide in the first amplificationproduct or a nucleotide of the 3′ end region of the first targetpolynucleotide complement in the first amplification product, and/or thesecond detector probe comprises a nucleotide of the 3′ end region of thesecond target polynucleotide in the second amplification product or anucleotide of the 3′ end region of the second target polynucleotidecomplement in the second amplification product.
 92. The method accordingto claim 88 wherein the first detector probe corresponds with anucleotide of a region upstream from the 3′ end region of the firsttarget polynucleotide in the first amplification product or a nucleotideof a region upstream from the 3′ end region of the first targetpolynucleotide complement in the first amplification product, and/or thesecond detector probe corresponds with a nucleotide of a region upstreamfrom the 3′ end region of the second target polynucleotide in the secondamplification product or a nucleotide of a region upstream from the 3′end region of the second target polynucleotide complement in the secondamplification product.
 93. The method according to claim 85 wherein thefirst target polynucleotide and/or the second target polynucleotide is18-25 ribonucleotides in length.
 94. The method according to claim 88wherein the first detector probe and/or second detector probe is a5′-nuclease cleavable probe.
 95. The method according to claim 94wherein the first detector probe and/or second detector probe comprisesFAM.
 96. The method according to claim 94 wherein the first detectorprobe and/or second detector probe comprises VIC.
 97. The methodaccording to claim 88 wherein the first detector probe and/or seconddetector probe comprises peptide nucleic acid (PNA).
 98. The methodaccording to claim 97 wherein first detector probe and/or seconddetector probe comprises FAM.
 99. The method according to claim 97wherein the first detector probe and/or second detector probe comprisesVIC.
 100. The method according to claim 88 wherein the first detectorprobe and/or the second detector probe comprises locked nucleic acid(LNA).
 101. The method according to claim 88 wherein the first detectorprobe and/or the second detector probe comprises a universal base. 102.The method according to claim 85 wherein the extending is a reversetranscription reaction comprising a reverse transcriptase.
 103. Themethod according to claim 85 wherein the stem of the first linker probeand/or the second linker probe comprises 12-16 base-pairs.
 104. Themethod according to claim 103 wherein the stem of the first linker probeand/or the second linker probe comprises 14 base-pairs.
 105. The methodaccording to claim 85 wherein the 3′ target specific portion of thefirst linker probe and/or the second linker probe comprises 5-8nucleotides.
 106. The method according to claim 85 wherein the loop ofthe first linker probe and/or the second linker probe comprises auniversal reverse primer portion.
 107. The method according to claim 85wherein the loop of the first linker probe and/or the second linkerprobe comprises 14-18 nucleotides.
 108. The method according to claim107 wherein the loop of the first linker probe and/or the second linkerprobe comprises 16 nucleotides.
 109. The method according to claim 88wherein the Tm of the first detector probe and/or second detector probeis 63-69 C.
 110. A method for detecting a miRNA molecule from a celllysate comprising; hybridizing the miRNA molecule from the cell lysatewith a linker probe, wherein the linker probe comprises a stem, a loop,and a 3′ target specific portion, wherein the 3′ target-specific portionbase pairs with the 3′ end region of the miRNA, extending the linkerprobe to form an extension reaction product; amplifying the extensionreaction product to form an amplification product in the presence of adetector probe, wherein the detector probe comprises a nucleotide of thelinker probe stem of the amplification product or a nucleotide of thelinker probe stem complement in the amplification product, and thedetector probe further comprises a nucleotide of the 3′ end region ofthe miRNA in the amplification product or a nucleotide of the 3′ endregion of the miRNA complement in the amplification product; and,detecting the miRNA molecule.
 111. The method according to claim 110,wherein the cell lysate comprises; treating cells with a lysis buffer,wherein the lysis buffer comprises, 10 mM Tris-HCl, pH 8.0; 0.02% SodiumAzide; and, 0.03% Tween-20.
 112. A kit comprising; a reversetranscriptase and a linker probe, wherein the linker probe comprises astem, a loop, and a 3′ target-specific portion, wherein the 3′target-specific portion corresponds to a miRNA.
 113. The kit accordingto claim 112 further comprising a DNA polymerase.
 114. The kit accordingto claim 112 further comprising a primer pair.
 115. The kit according toclaim 114 wherein the primer pair comprises, a forward primer specificfor a miRNA, and, a universal reverse primer, wherein the universalreverse primer comprises a nucleotide of the loop of the linker probe.116. The kit according to claim 112 comprising a plurality of primerpairs, wherein each primer pair is in one reaction vessel of a pluralityof reaction vessels.
 117. The kit according to claim 112 furthercomprising a detector probe.
 118. The kit according to claim 117 whereinthe detector probe comprises a nucleotide of the linker probe stem inthe amplification product or a nucleotide of the linker probe stemcomplement in the amplification product, and the detector probe furthercomprises a nucleotide of the 3′ end region of the miRNA in theamplification product or a nucleotide of the 3′ end region of the miRNAcomplement in the amplification product.